Corn plants and seed enhanced for asparagine and protein

ABSTRACT

The present invention relates to a corn plant and seed with enhanced levels of protein and amino acids. The invention also relates to DNA constructs that provide expression in transgenic corn cells of an asparagine synthetase enzyme. The DNA constructs are used in a method to produce transgenic corn plants and seeds and to select for plants and seeds with enhanced levels of protein and amino acids.

This application is a continuation of U.S. Ser. No. 12/694,152, filedJan. 26, 2010, now abandoned, which application is a divisional of U.S.Ser. No. 11/434,566, filed May 15, 2006, now abandoned, whichapplication claims the priority of U.S. Provisional Appl. Ser. No.60/681,348, filed May 16, 2005, the entire disclosures of which areincorporated herein by reference in their entireties. This applicationis also a continuation-in-part of U.S. Ser. No. 11/978,677, nowabandoned, filed Oct. 30, 2007, which application is acontinuation-in-part under 35 U.S.C. §120 of U.S. application Ser. No.10/425,114, filed on Apr. 28, 2003 (abandoned), which is herebyincorporated by reference in its entirety, including a copy of itsSequence Listing and Table 1. U.S. application Ser. No. 10/425,114, nowabandoned, claims priority under 35 U.S.C. §120 as acontinuation-in-part of U.S. application Ser. No. 09/826,019, filed Apr.5, 2001 (abandoned), which is a continuation-in-part of U.S. applicationSer. No. 09/773,370, filed Feb. 1, 2001 (abandoned), which claims thebenefit under 35 U.S.C. §119 to U.S. Provisional Application Ser. No.60/179,730, filed Feb. 2, 2000. U.S. application Ser. No. 10/425,114also claims priority under 35 U.S.C. §120 as a continuation-in-part ofU.S. application Ser. No. 10/219,999, filed Aug. 15, 2002 (abandoned),which claims the benefit under 35 U.S.C. §119 to U.S. ProvisionalApplication Ser. No. 60/324,109, filed Sep. 21, 2001, and to U.S.Provisional Application Ser. No. 60/312,544, filed Aug. 15, 2001. U.S.application Ser. No. 10/425,114 also claims priority under 35 U.S.C.§120 as a continuation-in-part of U.S. application Ser. No. 09/666,355,filed Sep. 20, 2000. U.S. application Ser. No. 10/425,114 also claimspriority under 35 U.S.C. §120 as a continuation-in-part of U.S.application Ser. No. 09/985,678, filed Nov. 5, 2001 (abandoned), whichis a continuation of U.S. application Ser. No. 09/304,517, filed May 6,1999 (abandoned). U.S. application Ser. No. 10/425,114 also claimspriority under 35 U.S.C. §120 as a continuation-in-part of U.S.application Ser. No. 09/849,529, filed May 7, 2001 (abandoned), whichclaims the benefit under 35 U.S.C. §119 to U.S. Provisional ApplicationSer. No. 60/196,868, filed May 9, 2000. U.S. application Ser. No.10/425,114 also claims priority under 35 U.S.C. §120 as acontinuation-in-part of U.S. application Ser. No. 09/804,730, filed Mar.13, 2001 (abandoned), which claims the benefit under 35 U.S.C. §119 toU.S. Provisional Application Ser. No. 60/189,657, filed Mar. 15, 2000.U.S. application Ser. No. 10/425,114 also claims priority under 35U.S.C. §120 as a continuation-in-part of U.S. application Ser. No.09/850,147, filed May 8, 2001 (abandoned), which claims the benefitunder 35 U.S.C. §119 to U.S. Provisional Application Ser. No.60/202,213, filed May 8, 2000. All of the foregoing applications arehereby incorporated by reference in their entirety, including theirrespective sequence listing and tables.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of plantbiotechnology and more specifically to enhancing asparagine and proteinin corn plants and seed.

2. Description of Related Art

Farmers and consumers desire crop plants with improved agronomic traitssuch as increased yield, increased seed protein production, and improvednutritional composition. Desirable nutritional components of crop plantsinclude, among others, fiber, antioxidants such as Vitamin E, selenium,iron, magnesium, zinc, B vitamins, lignans, phenolic acids, essentialamino acids, and phytoestrogens. Although considerable efforts in plantbreeding have provided some gains in these desired traits, the abilityto introduce specific non-host DNA into a plant genome provides furtheropportunities for generation of plants with these traits. In particular,while the yield of conventional corn has steadily increased over theyears, there has not been a similar increase in the capacity of cornplants to assimilate nitrogen more efficiently or to increase seedprotein content.

Availability of nitrogen has a significant positive impact on plantproductivity, biomass, and crop yield including the production of seedprotein. In plants, inorganic nitrogen is assimilated from the soil,reduced to ammonia, and incorporated into organic nitrogen in the formof the nitrogen-transporting amino acids asparagine, glutamine, asparticacid and glutamic acid. Asparagine (Asn) is the preferred amidetransport molecule because of its high nitrogen to carbon ratio (2N:4Cversus 2N:5C) and because it is relatively inert. Asn and other aminoacids are also used as building blocks for protein synthesis.

In plants, Asn is synthesized from glutamine, aspartate and ATP, in areaction catalyzed by the enzyme asparagine synthetase (AsnS).Glutamate, AMP and pyrophosphate are formed as by-products. Two forms ofAsnS have been described: a glutamine-dependent form and anammonia-dependent form. The glutamine-dependent AsnS can catalyze boththe glutamine-dependent and ammonia-dependent reactions althoughglutamine is the preferred nitrogen source.

High concentration of protein is considered an important quality traitfor most major crops, including soybean, corn, and wheat. Varieties ofhigh protein corn, wheat, and soybeans, for example, have beenidentified through traditional breeding. However, most of the highprotein lines developed this way have yield drag or other agronomicdisadvantages. It would be desirable if the protein content of crops,especially corn, could be increased above the presently availablelevels, both for human consumption and for use of the product in animalfeeds. This would offer the benefit of greatly enhanced nutrient valuewhen the crop is used as food and feed for humans and animals.

SUMMARY OF THE INVENTION

The present invention provides a method and compositions for treatmentof crops and other plant products so as to increase the protein andamino acid content in plants. The method and compositions increase thelevel of free amino acids and protein in corn tissues, particularly inseeds. More specifically, a transgenic corn plant and seed is providedthat contains in its genome a heterologous DNA composition thatexpresses a gene product involved in increased asparagine and increasedprotein biosynthesis. The expression of the product enhances thenutritional value of food corn and feed corn sources and processedproducts derived from the transgenic corn seed or parts thereof.

In one aspect, the invention provides methods for increasing proteincontent in a corn plant. A DNA construct comprising a polynucleotidesequence selected from the group consisting of SEQ ID NOs 1, 3, 5, 7, 9,10, 11, 12, 13, 14, 15, 16, and 17 wherein the polynucleotide moleculeencodes an asparagine synthetase polypeptide or polypeptide havingasparagine synthetase activity is also included.

In one embodiment, the present invention comprises a corn plant celltransformed with the heterologous DNA composition encoding an asparaginesynthetase identified as SEQ ID NO: 4. More specifically, the expressionof the heterologous corn AsnS2 (asparagine synthetase isozyme 2)polynucleotide molecule in the transgenic corn plant results in anelevated level of asparagine and protein in the transgenic plant, forexample, in the seeds of the corn plant compared to a corn plant of thesame variety not expressing the heterologous corn AsnS2 polynucleotidemolecule.

The present invention also relates to animal feed comprising theaforementioned seed with increased protein or amino acid content, or aprocessed product of such seed, for example, a meal. Accordingly, thepresent invention also encompasses a corn seed containing an asparaginesynthetase enzyme produced by expression of a heterologous DNA constructcomprising a DNA molecule encoding a corn asparagine synthetase enzyme.One embodiment of such a seed is harvested grain, the present inventionalso encompasses meal, gluten and other corn products made from suchgrain.

The present invention includes isolated nucleic acid primer sequencescomprising one or more of SEQ ID NOs 18-45, or the complement thereof.The present invention includes a method to detect or identify, in thegenome of a transformed plant or progeny thereof, a heterologouspolynucleotide molecule encoding a plant AsnS polypeptide, or a plantpolypeptide having AsnS activity of the present invention, comprising apolynucleotide molecule selected from the group consisting of SEQ ID NOs18-45, wherein said polynucleotide molecule is used as a DNA primer in aDNA amplification method.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the plasmid map of pMON79706.

FIG. 2 illustrates the plasmid map of pMON66229.

FIG. 3 illustrates the plasmid map of pMON66230.

FIG. 4 illustrates the plasmid map of pMON66231.

FIG. 5 illustrates the plasmid map of pMON66239.

FIG. 6 Transgene expression in pMON79706 events. Error bars represent95% confidence interval, with n=5 for transgenic events and n=10 forinbred control.

FIG. 7. Transgene expression in pMON92870 events. Error bars represent95% confidence interval, with n>3 plants for transgenic events and n=8plants for inbred control.

DESCRIPTION OF THE NUCLEIC ACID AND POLYPEPTIDE SEQUENCES

SEQ ID NO: 1 is a polynucleotide sequence encoding a Zea mays AsnS1.

SEQ ID NO: 2 is a Zea mays AsnS1 polypeptide.

SEQ ID NO: 3 is a polynucleotide sequence encoding a Zea mays AsnS2.

SEQ ID NO: 4 is a Zea mays AsnS2 polypeptide.

SEQ ID NO: 5 is a polynucleotide sequence encoding a Zea mays AsnS3.

SEQ ID NO: 6 is a Zea mays AsnS3 polypeptide.

SEQ ID NO: 7 is a polynucleotide sequence encoding a Glycine max AsnS.

SEQ ID NO: 8 is a Glycine max AsnS polypeptide.

SEQ ID NO: 9 is a polynucleotide sequence encoding a Xylella fastidiosaAsnS.

SEQ ID NO: 10 is a polynucleotide sequence encoding a Xanthomonascampestris AsnS.

SEQ ID NO: 11 is a polynucleotide sequence encoding a Bacillushalodurans AsnS.

SEQ ID NO: 12 is a polynucleotide sequence encoding an Oryza sativaAsnS.

SEQ ID NO: 13 is a polynucleotide sequence encoding a Galdieriasulphuraria AsnS.

SEQ ID NO: 14 is a polynucleotide sequence encoding a Galdieriasulphuraria AsnS.

SEQ ID NO: 15 is a polynucleotide sequence encoding a Galdieriasulphuraria AsnS.

SEQ ID NO: 16 is a polynucleotide sequence encoding a Galdieriasulphuraria AsnS.

SEQ ID NO: 17 is a polynucleotide sequence encoding a Saccharomycescerevisiae CGPG3913 AsnS.

SEQ ID NO: 18 is a forward (f) AsnS PCR primer sequence.

SEQ ID NO: 19 is a forward (f) AsnS PCR primer sequence.

SEQ ID NOs 20-43, are primary and secondary forward (f) and reverse (r)AsnS PCR primer sequences used in a Gateway cloning procedure.

SEQ ID NO: 44, a forward (f) AsnS PCR primer sequence.

SEQ ID NO: 45, a forward (f) AsnS PCR primer sequence.

SEQ ID NO:46 ZmASsense primer

SEQ ID NO:47 ZmASantisense primer

SEQ ID NO: 48 corn AsnS3 forward primer

SEQ ID NO: 49 corn AsnS3 reverse primer

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the invention provided to aidthose skilled in the art in practicing the present invention. Unlessotherwise defined herein, terms are to be understood according toconventional usage by those of ordinary skill in the relevant art.Definitions of common terms in molecular biology may also be found inRieger et al., 1991; and Lewin, 1994. The nomenclature for DNA bases asset forth at 37 CFR §1.822 is used. The standard one- and three-letternomenclature for amino acid residues is used. Modifications andvariations in the embodiments described herein may be made by those ofordinary skill in the art without departing from the spirit or scope ofthe present invention.

The present invention provides a method to increase protein content in acorn plant by introducing into the genome of a corn plant cell aheterologous polynucleotide that expresses an AsnS polypeptide in thetransgenic plant cell. The present invention provides DNA constructsthat comprise (comprise means “including but not limited to”)polynucleotide molecules, or segments of a polynucleotide molecule thatencode an AsnS polypeptide, optionally operably linked to a chloroplasttransit peptide.

Polynucleotide molecules encoding a AsnS polypeptide or analog or allelethereof, or polynucleotide molecules encoding a transit peptide ormarker/reporter gene are “isolated” in that they have been at leastpartially prepared in vitro, e.g., isolated from its native state, froma cell, purified, and amplified, e.g., they are in combination withgenetic elements heterologous to those found normally associated withthem in their native state. As used herein, a heterologous DNA constructcomprising an AsnS encoding polynucleotide molecule that has beenintroduced into a host cell, is preferably not identical to anypolynucleotide molecule present in the cell in its native, untransformedstate and is isolated with respect of other DNA molecules that occur inthe genome of the host cell.

As used herein, “altered or increased” levels of asparagine in atransformed plant, plant tissue, or plant cell are levels which aregreater than the levels found in the corresponding plant, plant tissue,or plant cells not containing the DNA constructs of the presentinvention.

The agents of the present invention may also be recombinant. As usedherein, the term recombinant means any agent (e.g., DNA, peptide, etc.),that is, or results, however indirectly, from human manipulation of apolynucleotide molecule.

As used herein in a preferred aspect, an increase in the nutritionalquality of a seed, for example, increased seed protein content, isdetermined by the ability of a plant to produce a seed having a higheryield of protein or a nutritional component than a seed without suchincrease in protein or nutritional quality. In a particularly preferredaspect of the present invention, the increase in nutritional quality ismeasured relative to a plant with a similar genetic background to thenutritionally enhanced plant except that the plant of the presentinvention expresses or over expresses a protein or fragment thereofdescribed in the heterologous DNA constructs herein.

Polynucleotide Molecules

The present invention includes and provides transgenic corn plants andseed that comprise in their genome a transgene comprising a heterologousDNA molecule encoding a corn asparagine synthetase (Zm.AsnS2) enzyme,the DNA molecule, for example, comprising SEQ ID NO: 3 and sequenceshaving at least 90%, 95%, or 99% identity to such sequences withfunctional asparagine synthetase activity.

A further aspect of the invention is a method for increasing protein ina corn plant by introducing into a corn cell a DNA construct thatprovides a heterologous polynucleotide molecule, for example, SEQ ID NOs1, 3, 5, 7, 9, 10, 11, 12, 13, 14, 15, 16 and 17 that encode anasparagine synthetase enzyme. The polynucleotide can differ from any ofthese examples without altering the polypeptide for which it encodes.For example, it is understood that codons capable of coding for suchconservative amino acid substitutions are known in the art.Additionally, the invention contemplates that polypeptides in which oneor more amino acid have been deleted, substituted, or added withoutaltering the asparagine synthetase function can be used in the invention

In one aspect of the present invention the polynucleotide of the presentinvention are said to be introduced polynucleotide molecules. Apolynucleotide molecule is said to be “introduced” if it is insertedinto a cell or organism as a result of human manipulation, no matter howindirect. Examples of introduced polynucleotide molecules include,without limitation, polynucleotides that have been introduced into cellsvia transformation, transfection, injection, and projection, and thosethat have been introduced into an organism via conjugation, endocytosis,phagocytosis, etc. Preferably, the polynucleotide is inserted into thegenome of the cell.

One subset of the polynucleotide molecules of the present invention isfragment polynucleotide molecules. Fragment polynucleotide molecules mayconsist of significant portion(s) of, or indeed most of, thepolynucleotide molecules of the present invention, such as thosespecifically disclosed. Alternatively, the fragments may comprisesmaller oligonucleotides (having from about 15 to about 400 nucleotideresidues and more preferably, about 15 to about 30 nucleotide residues,or about 50 to about 100 nucleotide residues, or about 100 to about 200nucleotide residues, or about 200 to about 400 nucleotide residues, orabout 275 to about 350 nucleotide residues). A fragment of one or moreof the polynucleotide molecules of the present invention may be a probeand specifically a PCR primer molecule. A PCR primer is a polynucleotidemolecule capable of initiating a polymerase activity while in adouble-stranded structure with another polynucleotide. Various methodsfor determining the structure of PCR probes and PCR techniques exist inthe art.

As used herein, two polynucleotide molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded polynucleotide structure.

A polynucleotide molecule is said to be the “complement” of anotherpolynucleotide molecule if they exhibit complete complementarity. Asused herein, molecules are said to exhibit “complete complementarity”when every nucleotide of one of the molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., (2001), and by Haymes etal., (1985). Departures from complete complementarity are thereforepermissible, as long as such departures do not completely preclude thecapacity of the molecules to form a double-stranded structure. Thus, inorder for a polynucleotide molecule to serve as a primer or probe itneed only be sufficiently complementary in sequence to be able to form astable double-stranded structure under the particular solvent and saltconcentrations employed.

Appropriate stringency conditions which promote DNA hybridization are,for example, 6.0×sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 20-25° C., are known to those skilledin the art or can be found in Ausubel, et al., eds. (1989), section6.3.1-6.3.6. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 65° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant such that a nucleic acidwill specifically hybridize to one or more of the polynucleotidemolecules provided herein, for example, as set forth in: SEQ ID NOs 1,3, 5, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18-45 and complementsthereof, under moderately stringent conditions, for example at about2.0×SSC and about 65° C.

In one embodiment of a method of the present invention, any of thepolynucleotide sequences or polypeptide sequences, or fragments ofeither, of the present invention can be used to search for relatedsequences. As used herein, “search for related sequences” means anymethod of determining relatedness between two sequences, including, butnot limited to, searches that compare sequence homology: for example, aPBLAST search of a database for relatedness to a single polypeptidesequence. Other searches may be conducted using profile based methods,such as the HMM (Hidden Markov model) META-MEME, which is maintained bySouth Dakota State University, SD, and PSI-BLAST, which is maintained bythe National Center for Biotechnology Information, National Library ofMedicine, National Institutes of Health (NCBI).

A polynucleotide molecule can encode for a substantially identical orsubstantially homologous polypeptide molecule. The degree of identity orhomology can be determined by use of computer software such as theWISCONSIN PACKAGE Gap Program. The Gap program in the WISCONSIN PACKAGEversion 10.0-UNIX from Genetics Computer Group, Inc. is based on themethod of Needleman and Wunsch, 1970. Using the TBLASTN program in theBLAST 2.2.1 software suite (Altschul et al., (1997, or using BLOSUM62matrix (Henikoff and Henikoff, 1992). A polynucleotide molecule of thepresent invention can also encode a homolog polypeptide. As used herein,a homolog polypeptide molecule or fragment thereof is a counterpartprotein molecule or fragment thereof in a second species (e.g., cornrubisco small subunit is a homolog of Arabidopsis rubisco smallsubunit). A homolog can also be generated by molecular evolution or DNAshuffling techniques, so that the molecule retains at least onefunctional or structure characteristic of the original polypeptide (see,for example, U.S. Pat. No. 5,811,238).

In a preferred embodiment, any of the polynucleotide molecules of thepresent invention can be operably linked to a promoter region thatfunctions in a plant cell to cause the production of an mRNA molecule,where the polynucleotide molecule that is linked to the promoter isheterologous with respect to that promoter. As used herein,“heterologous” DNA is any DNA sequence which is not naturally found nextto the adjacent DNA. “Native” refers to a naturally occurring nucleicacid sequence. “Heterologous” sequence often originates from a foreignsource or species or, if from the same source, is modified from itsoriginal form and/or location in the genome.

As used herein, the terms “protein,” “peptide molecule,” or“polypeptide” includes any molecule that comprises five or more aminoacids. It is well known in the art that protein, peptide, or polypeptidemolecules may undergo modification, including post-translationalmodifications, such as, but not limited to, disulfide bond formation,glycosylation, phosphorylation, or oligomerization. Thus, as usedherein, the terms “protein,” “peptide molecule,” or “polypeptide”includes any protein that is modified by any biological ornon-biological process. The terms “amino acid” and “amino acids” referto all naturally occurring L-amino acids. This definition is meant toinclude norleucine, norvaline, ornithine, homocysteine, and homoserine.

A “protein fragment” is a peptide or polypeptide molecule whose aminoacid sequence comprises a subset of the amino acid sequence of thatprotein. A protein or fragment thereof that comprises one or moreadditional peptide regions not derived from that protein is a “fusion”protein. Such molecules may be derivatized to contain carbohydrate orother moieties (such as keyhole limpet hemocyanin). Fusion protein orpeptide molecules of the present invention are preferably produced viarecombinant means.

Plant Constructs and Plant Transformants

One or more of the DNA constructs of the present invention that encodefor an asparagine synthetase may be used in plant transformation ortransfection. Exogenous genetic material may be transferred into a plantcell and the plant cell regenerated into a whole, fertile, or sterileplant. Exogenous genetic material is any genetic material, whethernaturally occurring or otherwise, from any source that is capable ofbeing inserted into any organism.

In a further aspect of the present invention, polynucleotide sequencesof the present invention also encode peptides involved in intracellularlocalization, export, or post-translational modification, for examplechloroplast transit peptides.

As used herein, the term “gene” includes a nucleic acid molecule thatprovides regulation of transcription that includes a promoter thatfunctions in plants, 5′ untranslated molecules, e.g., introns and leadersequences, a transcribed nucleic acid molecule and a 3′ transcriptionaltermination molecule.

The polynucleic acid molecules encoding a polypeptide of the presentinvention may be combined with other non-native, or heterologoussequences in a variety of ways. By “heterologous” sequences it is meantany sequence that is not naturally found joined to the nucleotidesequence encoding polypeptide of the present invention, including, forexample, combinations of nucleotide sequences from the same plant thatare not naturally found joined together, or the two sequences originatefrom two different species. The term “operably linked”, as used inreference to the physical and function arrangement of regulatory andstructural polynucleotide molecules that causes regulated expression ofan operably linked structural polynucleotide molecule.

The expression of a DNA construct or transgene means the transcriptionand stable accumulation of sense or antisense RNA or protein derivedfrom the polynucleotide molecule of the present invention or translationthereof. “Sense” RNA means RNA transcript that includes the mRNA and socan be translated into polypeptide or protein by the cell. “AntisenseRNA” means a RNA transcript that is complementary to all or part of atarget primary transcript or mRNA and that blocks the expression of atarget gene (U.S. Pat. No. 5,107,065, incorporated herein by reference).The complementarity of an antisense RNA may be with any part of thespecific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-translated sequence, introns, or the coding sequence. “RNAtranscript” means the product resulting from RNA polymerase-catalyzedtranscription of a DNA sequence. When the RNA transcript is a perfectcomplementary copy of the DNA sequence, it is referred to as the primarytranscript or it may be a RNA sequence derived from post-transcriptionalprocessing of the primary transcript and is referred to as the matureRNA.

As used herein, the term plant expression cassette refers to a constructcomprising the necessary DNA regulatory molecules operably linked to thetarget molecule to provide expression in a plant cell.

The DNA construct of the present invention can, in one embodiment,contain a promoter that causes the over expression of the polypeptide ofthe present invention, where “overexpression” means the expression of apolypeptide either not normally present in the host cell, or present insaid host cell at a higher level than that normally expressed from theendogenous gene encoding said polypeptide. Promoters, which can causethe overexpression of the polypeptide of the present invention, aregenerally known in the art, examples of such that provide constitutiveexpression pattern include cauliflower mosaic virus 19S promoter andcauliflower mosaic virus 35S promoter (U.S. Pat. No. 5,352,605), figwortmosaic virus 35S promoter (U.S. Pat. No. 6,051,753), sugarcanebacilliform virus promoter (U.S. Pat. No. 5,994,123), commelina yellowmottle virus promoter (Medberry et al., 1992), small subunit ofribulose-1,5-bisphosphate carboxylase promoter, rice cytosolictriosephosphate isomerase promoter, adenine phosphoribosyltransferasepromoter, rice actin 1 promoter (U.S. Pat. No. 5,641,876), maizeubiquitin promoter, mannopine synthase promoter and octopine synthasepromoter.

Such genetic constructs may be transferred into either monocotyledonousor dicotyledonous plants including, but not limited to alfalfa, apple,Arabidopsis, banana, Brassica campestris, canola, castor bean, coffee,corn, cotton, cottonseed, chrysanthemum, crambe, cucumber, Dendrobiumspp., Dioscorea spp., eucalyptus, fescue, flax, gladiolus, liliacea,linseed, millet, muskmelon, mustard, oat, oil palms, oilseed rape,peanut, perennial ryegrass, Phaseolus, rapeseed, rice, sorghum, soybean,rye, tritordeum, turfgrass, wheat, safflower, sesame, sugarbeet,sugarcane, cranberry, papaya, safflower, and sunflower (Christou, 1996).In a preferred embodiment, the genetic material is transferred into acorn cell.

Transfer of a polynucleotide molecule that encodes a protein can resultin expression or overexpression of that polypeptide in a transformedcell or transgenic plant. One or more of the proteins or fragmentsthereof encoded by polynucleotide molecules of the present invention maybe overexpressed in a transformed cell or transformed plant.

In one embodiment, DNA constructs of the present invention comprise apolynucleotide molecule encoding a polypeptide sequence selected fromthe group consisting of SEQ ID NOs 1, 3, 5, 7, 9, 10, 11, 12, 13, 14,15, 16 and 17. The invention provides transformed corn cells wherein,relative to an untransformed corn plant without such a DNA construct,the cell has an enhanced asparagine level.

In another embodiment, DNA constructs of the present invention comprisea heterologous DNA molecule operably linked to a corn asparaginesynthetase coding sequence, for example, SEQ ID NOs 1, 3, or 5, and theDNA construct is transformed corn cell In a preferred embodiment, DNAconstructs of the present invention comprising SEQ ID NO: 3 are providedin a transformed corn cell, and expression of the DNA construct providesa corn plant tissue with increased asparagine or a corn plant seed withincreased protein relative to a corn plant not transformed with the DNAconstruct.

In some embodiments, the levels of one or more products of the AsnS maybe increased throughout a plant or localized in one or more specificorgans or tissues of the plant. Without limiting the scope of thepresent invention, several promoter sequences are useful for expressingthe gene of the above enzyme. For example, maize C4 type PPDK promoter(Glackin et al., 1990), maize C4 type PEPC promoter (Hudspeth and Grula,1989), rice Rubisco small subunit promoter (Kyozuka et al., 1993), andlight-harvesting chlorophyll a/b binding protein promoter (Sakamoto etal., 1991), the P-FDA promoter (US20040216189A1, the polynucleotidesequence of which is herein incorporated by reference) and P-RTBVpromoter (U.S. Pat. No. 5,824,857, the polynucleotide sequence of whichis herein incorporated by reference). For example the levels ofasparagine or protein may be increased in one or more of the tissues andorgans of a plant including without limitation: roots, tubers, stems,leaves, stalks, fruit, berries, nuts, bark, pods, seeds, and flowers. Apreferred organ is a seed.

For the purpose of expression in source tissues of the plant, such asthe leaf, seed, root, or stem, it is preferred that the promotersutilized have relatively high expression in these specific tissues.Tissue-specific expression of a protein of the present invention is aparticularly preferred embodiment.

DNA constructs or vectors may also include, with the coding region ofinterest, a polynucleotide sequence that acts, in whole or in part, toterminate transcription of that region. A number of such sequences havebeen isolated, including the T-NOS 3′ region (Ingelbrecht et al., 1989;Bevan et al., 1983). Regulatory transcript termination regions can beprovided in plant expression constructs of this present invention aswell. Transcript termination regions can be provided by the DNA sequenceencoding the gene of interest or a convenient transcription terminationregion derived from a different gene source, for example, the transcripttermination region that is naturally associated with the transcriptinitiation region. The skilled artisan will recognize that anyconvenient transcript termination region that is capable of terminatingtranscription in a plant cell can be employed in the constructs of thepresent invention.

A vector or construct may also include regulatory elements, such asintrons. Examples of such include, the Adh intron 1 (Callis et al.,1987), the sucrose synthase intron (Vasil et al., 1989), hsp70 intron(U.S. Pat. No. 5,859,347), and the TMV omega element (Gallie et al.,1989). These and other regulatory elements may be included whenappropriate.

A vector or construct may also include a selectable marker. Selectablemarkers may also be used to select for plants or plant cells thatcontain the exogenous genetic material. Examples of such include, butare not limited to: a neo gene (Potrykus et al., 1985), which codes forkanamycin resistance and can be selected for using kanamycin, nptII,G418, hpt, etc.; a bar gene, which codes for bialaphos resistance; amutant EPSP synthase gene (Hinchee et al., 1988; Reynaerts et al., 1988;Jones et al., 1987), which encodes glyphosate resistance; a nitrilasegene which confers resistance to bromoxynil (Stalker et al., 1988); amutant acetolactate synthase gene (ALS) which confers imidazolinone orsulphonylurea resistance (U.S. Pat. No. 4,761,373); D'Halluin et al.,1992); and a methotrexate resistant DHFR gene (Thillet et al., 1988).

Plant Transformation

The most commonly used methods for transformation of plant cells are theAgrobacterium-mediated DNA transfer process and the biolistics ormicroprojectile bombardment mediated process (i.e., the gene gun).Typically, nuclear transformation is desired but if it is desirable tospecifically transform plastids, such as chloroplasts or amyloplasts,plant plastids may be transformed utilizing a microprojectile-mediateddelivery of the desired polynucleotide.

The methods for introducing transgenes into plants byAgrobacterium-mediated transformation utilize a T-DNA (transfer DNA)that incorporates the genetic elements of the transgene and transfersthose genetic elements into the genome of a plant. Generally, thetransgene(s) bordered by a right border DNA molecule (RB) and a leftborder DNA molecule (LB) is (are) transferred into the plant genome at asingle locus. The “T-DNA molecule” refers to a DNA molecule thatintegrates into a plant genome via an Agrobacterium mediatedtransformation method. The ends of the T-DNA molecule are defined in thepresent invention as being flanked by the border regions of the T-DNAfrom Agrobacterium Ti plasmids. These border regions are generallyreferred to as the Right border (RB) and Left border (LB) regions andexist as variations in nucleotide sequence and length depending onwhether they are derived from nopaline or octopine producing strains ofAgrobacterium. The border regions commonly used in DNA constructsdesigned for transferring transgenes into plants are often severalhundred polynucleotides in length and comprise a nick site where anendonuclease digests the DNA to provide a site for insertion into thegenome of a plant. T-DNA molecules generally contain one or more plantexpression cassettes.

With respect to microprojectile bombardment (U.S. Pat. Nos. 5,550,318;5,538,880; and 5,610,042; each of which is specifically incorporatedherein by reference in its entirety), particles are coated withpolynucleotides and delivered into cells by a propelling force.Exemplary particles include those comprised of tungsten, platinum, andpreferably, gold. A useful method for delivering DNA into plant cells byparticle acceleration is the Biolistics Particle Delivery System(BioRad, Hercules, Calif.), which can be used to propel particles coatedwith DNA or cells through a screen, such as a stainless steel or Nytexscreen, onto a filter surface covered with monocot plant cells culturedin suspension. Microprojectile bombardment techniques are widelyapplicable, and may be used to transform virtually any plant species.Examples of species that have been transformed by microprojectilebombardment include monocot species such as corn (PCT Publication WO95/06128), barley, wheat (U.S. Pat. No. 5,563,055, incorporated hereinby reference in its entirety), rice, oat, rye, sugarcane, and sorghum;as well as a number of dicots including tobacco, soybean (U.S. Pat. No.5,322,783, incorporated herein by reference in its entirety), sunflower,peanut, cotton, tomato, and legumes in general (U.S. Pat. No. 5,563,055,incorporated herein by reference in its entirety).

To select or score for transformed plant cells regardless oftransformation methodology, the DNA introduced into the cell contains agene that functions in a regenerable plant tissue to produce a compoundthat confers upon the plant tissue resistance to an otherwise toxiccompound. Genes of interest for use as a selectable, screenable, orscorable marker would include but are not limited to GUS, greenfluorescent protein (GFP), luciferase (LUX), and antibiotic or herbicidetolerance genes. Examples of antibiotic resistance genes include thoseconferring resistance to kanamycin (and neomycin, G418), and bleomycin.

The regeneration, development, and cultivation of plants from varioustransformed explants are well documented in the art. This regenerationand growth process typically includes the steps of selecting transformedcells and culturing those individualized cells through the usual stagesof embryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil. Cells that survive the exposure to the selectiveagent, or cells that have been scored positive in a screening assay, maybe cultured in media that supports regeneration of plants. Developingplantlets are transferred to soil-less plant growth mix, and hardenedoff, prior to transfer to a greenhouse or growth chamber for maturation.

The present invention can be used with any transformable cell or tissue.By transformable as used herein is meant a cell or tissue that iscapable of further propagation to give rise to a plant. Those of skillin the art recognize that a number of plant cells or tissues aretransformable in which after insertion of exogenous DNA and appropriateculture conditions the plant cells or tissues can form into adifferentiated plant. Tissue suitable for these purposes can include butis not limited to immature embryos, scutellar tissue, suspension cellcultures, immature inflorescence, shoot meristem, nodal explants, callustissue, hypocotyl tissue, cotyledons, roots, and leaves.

Any suitable plant culture medium can be used. Examples of suitablemedia would include but are not limited to MS-based media (Murashige andSkoog, 1962) or N6-based media (Chu et al., 18:659, 1975) supplementedwith additional plant growth regulators including but not limited toauxins, cytokinins, ABA, and gibberellins. Those of skill in the art arefamiliar with the variety of tissue culture media, which whensupplemented appropriately, support plant tissue growth and developmentand are suitable for plant transformation and regeneration. These tissueculture media can either be purchased as a commercial preparation, orcustom prepared and modified. Those of skill in the art are aware thatmedia and media supplements such as nutrients and growth regulators foruse in transformation and regeneration and other culture conditions suchas light intensity during incubation, pH, and incubation temperaturesthat can be optimized for the particular variety of interest.

Any of the polynucleotide molecules of the present invention may beintroduced into a plant cell in a permanent or transient manner incombination with other genetic elements such as vectors, promoters,enhancers, etc. Further, any of the polynucleotide molecules of thepresent invention may be introduced into a plant cell in a manner thatallows for expression or overexpression of the protein or fragmentthereof encoded by the polynucleotide molecule.

The present invention also provides for parts of the plants,particularly reproductive or storage parts, of the present invention.Plant parts, without limitation, include seed, endosperm, ovule, pollen,or tubers. In a particularly preferred embodiment of the presentinvention, the plant part is a corn seed. In one embodiment the cornseed (or grain) is a constituent of animal feed.

In a preferred embodiment the corn feed or corn meal or protein from thecorn seed is designed for livestock animals or humans, or both. Methodsto produce feed, meal, and protein, are known in the art. See, forexample, U.S. Pat. Nos. 4,957,748; 5,100,679; 5,219,596; 5,936,069;6,005,076; 6,146,669; and 6,156,227. In a preferred embodiment, theprotein preparation is a high protein preparation. Such a high proteinpreparation preferably has a protein content of greater than about 5%(w/v), more preferably 10% (w/v), and even more preferably 15% (w/v).

Descriptions of breeding methods that are commonly used for differenttraits and crops can be found in one of several reference books (e.g.,Hayward, 1993; Richards, 1997; Allard, 1999).

Other Organisms

A polynucleotide of the present invention may be introduced into anycell or organism such as a mammalian cell, mammal, fish cell, fish, birdcell, bird, algae cell, algae, fungal cell, fungi, or bacterial cell. Aprotein of the present invention may be produced in an appropriate cellor organism. Preferred host and transformants include: fungal cells suchas Aspergillus, yeasts, mammals, particularly bovine and porcine,insects, bacteria, and algae. Particularly preferred bacteria areAgrobacterium tumefaciens and E. coli.

In an aspect of the present invention, one or more of the nucleic acidmolecules of the present invention are used to determine the level ofexpression (i.e., the concentration of mRNA in a sample, etc.) in aplant (preferably canola, corn, Brassica campestris, oilseed rape,rapeseed, soybean, crambe, mustard, castor bean, peanut, sesame,cottonseed, linseed, safflower, oil palm, flax or sunflower) or pattern(i.e., the kinetics of expression, rate of decomposition, stabilityprofile, etc.) of the expression of a protein encoded in part or wholeby one or more of the polynucleotide molecule of the present invention.A number of methods can be used to compare the expression between two ormore samples of cells or tissue. These methods include hybridizationassays, such as northerns, RNAase protection assays, and in situhybridization. Alternatively, the methods include PCR-type assays. In apreferred method, expression is assessed by hybridizing polynucleotidesfrom the two or more samples to an array of polynucleotides. The arraycontains a plurality of suspected sequences known or suspected of beingpresent in the cells or tissue of the samples.

The following examples are included to demonstrate aspects of theinvention, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificaspects which are disclosed and still obtain a like or similar resultwithout departing from the spirit and scope of the invention.

EXAMPLES

Those of skill in the art will appreciate the many advantages of themethods and compositions provided by the present invention. Thefollowing examples are included to demonstrate the preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. All references cited herein are incorporated herein byreference to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, or compositionsemployed herein.

Example 1 Construction of Corn and Soy Plant cDNA and Genomic Libraries

This example describes the production of cDNA libraries made from cornand soy plant tissues from which the corn AsnS and soy polynucleotidesequences of the present invention were isolated. cDNA Libraries weregenerated from Zea mays and Glycine max tissue using techniques known inthe art, for example, Alba, 2004. Corn cDNA libraries were made from twodifferent tissues. A library was made from incipient kernels harvestedat the dilatory phase from inbred line 90DDD5. A second corn cDNAlibrary was made from silk tissue at the silking growth stage from corninbred line H99 and germinating pollen from corn inbred line MO17. Forconstruction of a cDNA library from soybean (Glycine max), meristematictissue and part of the hypocotyl were excised from rehydrated drysoybean seeds of variety A3237 (Asgrow). Explants were prepared by firstgerminating surface sterilized seeds on solid tissue culture media for 6days at 28° C. at 18 hours of light/day, and then transferringgerminated seeds to 4° C. for at least 24 hours. For the tissue used inlibrary preparation the cotyledons were removed to enrich for thespecific tissue of interest. 0.5 to 2 grams of tissue were used forpreparation of total RNA and poly A+ RNA. For all cDNA libraries, planttissues were harvested and immediately frozen in liquid nitrogen. Theharvested tissue was stored at −80° C. until preparation of total RNA.The total RNA was purified using Trizol reagent from InvitrogenCorporation (Invitrogen Corporation, Carlsbad, Calif., U.S.A.),essentially as recommended by the manufacturer. Poly A+ RNA (mRNA) waspurified using magnetic oligo dT beads essentially as recommended by themanufacturer (Dynabeads, Dynal Biotech, Oslo, Norway).

Construction of plant cDNA libraries is well known in the art and anumber of cloning strategies exist. A number of cDNA libraryconstruction kits are commercially available. cDNA libraries wereprepared using the Superscript™ Plasmid System for cDNA synthesis andPlasmid Cloning (Invitrogen Corporation), as described in theSuperscript II cDNA library synthesis protocol. The cDNA libraries werechecked to confirm an appropriate insert:vector ratio.

A genomic DNA library was constructed using genomic DNA isolated fromZea mays using a modified genomic DNA isolation protocol described below(Dellaporta et al., 1983). Corn seedlings were grown in soil or in Petriplates, were harvested, and kept frozen in liquid nitrogen untilextraction. The tissue was ground to a fine powder using a mortar andpestle while keeping the tissue frozen with liquid nitrogen. Thepowdered tissue was transferred to a Waring blender containing 200 mL ofcold (0° C.) DNA extraction buffer (350 mM sorbitol; 100 mM Tris; 5 mMEDTA; pH to 7.5 with HCl; sodium bisulfite, 3.8 mg/mL) that was addedjust before use, and homogenized at high speed for 30-60 seconds. Thehomogenate was filtered through a layer of cheesecloth and collected ina centrifuge bottle. The samples were then centrifuged at 2500×g for 20minutes, and the supernatant and any loose green material werediscarded. The pellet was then resuspended in 1.25 mL of DNA extractionbuffer and transferred to a 50 mL polypropylene tube. Nuclei lysisbuffer (1.75 mL containing 200 mM Tris; 50 mM EDTA; 2 M NaCl; 2.0% (w/v)CTAB; pH adjusted to 7.5 with HCl) was then added, followed by additionof 0.6 mL of 5% (w/v) sarkosyl. The tubes were mixed gently, and thesamples were incubated at 65° C. for 20 minutes. An equal volume ofchloroform:isoamyl alcohol (24:1) was added and the tubes were againmixed gently. The tubes were then centrifuged at 2500×g for 15 minutes,and the resulting supernatant was transferred to a clean tube. An equalvolume of ice-cold isopropanol was poured onto the sample, and thesample was inverted several times until a precipitate formed. Theprecipitate was removed from the solution using a glass pipette andresidual alcohol removed by allowing the precipitate to air dry for 2-5minutes. The precipitate was resuspended in 400 μL TE buffer (10 mMTris-HCl, 1 mM EDTA, pH adjusted to 8.0).

Example 2 Isolation of AsnS Polynucleotide Sequences by LigationIndependent and Gateway Cloning Methods and Corn Transformation

This example illustrates the isolation of polynucleotide moleculesencoding AsnS using ligation independent and Gateway® cloning methodsand the construction of DNA constructs of the present invention thatcomprise the polynucleotide molecules that encode AsnS polypeptidesisolated from various plant and microorganisms sources as described inTable 1. The promoter molecules used to drive the expression of thelinked AsnS-encoding polynucleotide molecules are the rice actin 1promoter, P-Os.Act1 (U.S. Pat. No. 5,641,876, herein incorporated byreference); the Zea mays PPDK (Matsuoka et al., 1993), P-RTBV-1 (U.S.Pat. No. 5,824,857, herein incorporated by reference), and the P-Zm. NAS(promoter molecule of the genomic region coding for a nicotianaminesynthase 2 polypeptide from corn).

TABLE 1 AsnS coding sequence source, promoter and DNA constructs SEQ IDExemplary DNA NO: Coding sequence source Promoter construct 3 Zea maysAsnS2 P-Os.Act1 pMON79706 5 Zea mays AsnS3 P-Os.Act1 pMON92870 7 Glycinemax P-Os.Act1 pMON79700 17 Saccharomyces cerevisiae P-Os.Act1 PMON79653

Ligation independent cloning was developed to clone PCR products and isbased on the annealing of non-palindromic single-stranded ends. LIC isan efficient cloning method, which is not limited by restriction sitesor the need for restriction enzyme digestion or ligation reactions andleaves seamless junctions (Aslanidis and de Jong, 1990).

Terminal, single-stranded DNA segments are produced in the vectorthrough the use of a “nicking endonuclease” and restrictionendonuclease. A nicking endonuclease is an endonuclease that nicks onestrand of the polynucleotide duplex to create single stranded tails onthe cloning vector. The vector is first linearized with a standardrestriction endonuclease. This is then followed by digestion with anicking endonuclease. After heat treatment, terminal, single-strandedDNA segments are produced in the vector. A GC content of roughly 55% isrecommended for downstream PCR amplification and efficient annealing.The promoter, tag, or other sequence element can be added to the 5′ and3′ ends of the PCR-amplified product to create a linear construct thatcan be used in downstream applications.

The DNA construct pMON92870 was assembled from the base vector,pMON82060, and a corn AsnS3 polynucleotide molecule encoding an AsnSpolypeptide provided as SEQ ID NO 5. The plasmid backbone pMON82060 waslinearized using the restriction endonuclease, HpaI. The plasmidbackbone was then treated with the nicking endonuclease, N.BbvC IA (NewEngland Biolabs, Beverly, Mass.). After digestion, the reaction washeated to 65° C. This causes the nicked strands of DNA to disassociatefrom their complementary DNA strands. The resulting linearized plasmidbackbone was left with two terminal, single-stranded DNA segmentsavailable for assembly.

The polymerase chain reaction was employed to produce the terminalsingle-stranded DNA segments in the DNA molecule encoding AsnS. The cornAsnS3 polynucleotide sequence (SEQ ID NO: 5) encoding the AsnSpolypeptide was used for the design of the forward PCR primer (SEQ IDNO: 48) and the reverse PCR primer (SEQ ID NO: 49):

SEQ ID NO: 48: GCAGTCGCTGTCGTTACCCGGCATCATGTGGCATC SEQ ID NO: 49:GCGAGTACCGCTGGGTTCTAACGTACTCTCGTCAGACCGCGPolymerase chain reaction amplification was performed using the highfidelity thermal polymerase, KOD hot start DNA polymerase (Novagen,Madison, Wis.). The polymerase chain reaction was performed in a 25 μLvolume containing, 1×KOD hot start DNA polymerase buffer, 1M betaine(Sigma, St. Louis, Mo.), 1 mM MgSO4, 250 μM dNTPs, 5 pmols of eachprimer and 1 unit of KOD hot start DNA polymerase. The polymerase chainreaction was performed in a PTC-225 DNA Engine Tetrad™ thermal cycler(MJ Research Inc., Waltham, Mass.) using the following cyclerparameters:

-   1. 94° C. for 2 minutes-   2. 94° C. for 15 seconds-   3. 70° C. for 30 seconds (−1° C. per cycle)-   4. 72° C. for 5 minutes-   5. Go to step 2, 9 times-   6. 94° C. for 15 seconds-   7. 60° C. for 30 seconds-   8. 72° C. for 5 minutes-   9. Go to step 6, 24 times-   10. 72° C. for 10 minutes-   11. 10° C. hold-   12. end

A second round of polymerase chain reaction was performed to introduceuridine residues in the region in which the terminal, single-strandedDNA segments were produced. Many DNA polymerases are unable to readuridine residues in the template strand of DNA or are unable topolymerize strands using uridine residues. Polymerase chain reaction wastherefore performed using an enzyme capable of incorporating and readinguridines (Expand High Fidelity™ plus PCR System; Roche, Indianapolis,Ind.). Modification of this method and use of other methods that providethe expected result are known by those skilled in the art.

The assembled DNA construct was transformed into ElectroMAX™ DH10B E.coli competent cells (Invitrogen, Carlsbad, Calif.). A 0.54 (microliter)aliquot from the assembly reaction was mixed with 20 μL of ElectroMAX™DH10B competent cells on ice and loaded into a MicroPulser 0.2 mmelectroporation cuvette (Bio-Rad Laboratories Inc., Hercules Calif.) forelectroporation. Cells were subjected to electroporation at 1.8 kV usinga 165-2100 MicroPulser Electroporator (Bio-Rad Laboratories Inc.).Electroporated cells were incubated in 180 μL of SOC medium (InvitrogenInc.) at 37° C. for 1 hour. Cells were then plated onto LB agar platescontaining spectinomycin (75 mg/L) and grown overnight at 37° C.Colonies were selected and grown in LB media overnight at 37° C. Theplasmid DNA construct was isolated using the QIAprep® Spin Miniprep Kit(QIAgen Sciences, Valencia, Calif.). DNA sequencing was performed on anABI 3730×1 DNA Analyzer, using BigDye® terminator (Applied Biosystems,Foster City, Calif.).

The cloning of corn AsnS2, soy AsnS, and yeast AsnS1 AsnS-encodingpolynucleotide sequences was accomplished using the “Gateway® cloningmethod” as described by the manufacturer (Invitrogen Corp.). The goal ofthe Gateway® cloning method is to make an expression clone. Thistwo-step process involves first, the cloning of the gene of interestinto an entry vector, followed by subcloning of the gene of interestfrom the entry vector into a destination vector to produce an expressionvector. The cloning technology is based on the site-specificrecombination system used by phage lambda to integrate its DNA into theE. coli chromosome.

DNA constructs for use in subsequent recombination cloning, two attB orattR recombination sequences were cloned into a recombinant vectorflanking a Spectinomycin/Streptomycin resistance gene (SPC/STR) and anAsnS-encoding polynucleotide sequence. The AsnS-encoding polynucleotidesequences were isolated from cDNA or genomic libraries made from theirrespective species using the primary and secondary primer sequences (SEQID NOs 20-43). The contiguous attB1/R1, SPC/STR gene, AsnS gene, andattB2/R2 sequences were moved as a single polynucleotide molecule into arecombinant construct for expression in plant cells, the double-strandedDNA plasmids designated pMON79706 (Zea mays AsnS2), pMON79700 (Glycinemax AsnS) or pMON79653 (Saccharomyces cerevisiae AsnS). These DNAconstructs comprise the Agrobacterium right border (O-OTH.-RB) regionsand left border (LB) regions, and others disclosed by Herrera-Estrellaet al., 1983; Bevan, 1984; Klee et al., 1985, the e35S promoter (P-CAMV.35S, tandemly duplicated enhancer U.S. Pat. No. 5,322,938), the attB1/R1genetic element (O-Lam. attB1/R1), the SPC/STR gene, the respectiveAsnS-coding region (CR), the attB2/R2 genetic element (O-Lam.attB2/R2),the potato protease inhibitor II terminator (St.Pis), the AgrobacteriumNOS promoter (P-AGRtu..nos, Fraley et al., 1983), the Agrobacterium leftborder (O-OTH.-LB), the kanamycin resistance gene (CR-OTH.-Kan, U.S.Pat. No. 6,255,560), and the E. coli origin of replication(Ec.ori.ColE).

The DNA constructs were amplified in Library Efficiency® DB3.1™ cells(Invitrogen Corporation) under chloramphenicol selection (25 μg/mL) andkanamycin selection (50 μg/mL) for pMON79706, pMON79700 or pMON79653.Vector DNA was purified from bacterial cultures using a QIAGEN PlasmidKit (QIAGEN Inc.).

DNA for pMON79700, pMON79706, and pMON79653 was introduced into the cornembryos as described in U.S. Pat. No. 5,015,580, using the electricdischarge particle acceleration gene delivery device. Formicroprojectile bombardment of LH59 pre-cultured immature embryos, 35%to 45% of maximum voltage was preferably used. Following microprojectilebombardment, the corn tissue was cultured in the dark at 27° C.Transformation methods and materials for making transgenic plants ofthis invention, for example, various media and recipient target cells,transformation of immature embryos and subsequent regeneration offertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and6,232,526 and U.S. Patent Application Publication 20040216189, which areincorporated herein by reference.

Fertile transgenic corn plants were produced from transformed corn cellsby growing transformed callus on the appropriate regeneration media toinitiate shoot development and plantlet formation. Plantlets weretransferred to soil when they were about 3 inches tall and possessedroots (about four to 6 weeks after transfer to medium). Plants weremaintained for two weeks in a growth chamber at 26° C., followed by twoweeks on a mist bench in a greenhouse. The plants were subsequentlytransplanted into 5-gallon pots and grown to maturity in the greenhouse.Reciprocal pollinations were made with the corn LH59 inbred line. Seedwas collected from corn plants and used for analysis of protein andfurther breeding activities.

Example 3 Vector Construction and Transformation of Corn with AsnSPolynucleotide Sequences

The corn AsnS2 (SEQ ID NO: 3, pMON79706, FIG. 1) was amplified by use ofPCR (polymerase chain reaction). The reaction conditions for the PCRreaction followed the manufacturer's protocol (PE Applied Biosystems,Foster City, Calif.). Approximately 100 ng of corn DNA, prepared asdescribed above, was amplified using 30 nmole each of forward (f) primer(SEQ ID NO: 32) and reverse (r) primer (SEQ ID NO: 33) and 10 micromoleseach of dATP, dCTP, dGTP and TTP, 2.5 units of TaKaRaLA Taq in 1× LA PCRBuffer II (Takara Bio INC, Shiga, Japan). After initial incubation at94° C. for 1 minute, 35 cycles of PCR were performed at 94° C. for 45seconds, followed by annealing at 60° C. for 45 seconds, 72° C. for 1minute 15 seconds, followed, by 1 cycle of 72° C. for 7 minutes.

Five AsnS2 DNA constructs were made. The first corn AsnS2 construct wasmade by isolating an 1821 base pair AsnS2 fragment from pMON79706 byPCR, as described above, followed by restriction digestion with XbaI andEcoRI restriction enzymes. The resulting AsnS2 gene was ligated intopMON61560, which had also been digested with XbaI and EcoRI. Theresulting shuttle vector (pMON66246) was digested with NotI and theinsert containing the AsnS2 gene, in operable linkage with the PPDKpromoter and RGLUT1 terminator, was ligated into pMON30167, which hadalso been digested with NotI. The pMON30167 plasmid, which contains theEPSPS gene, provides for selection with glyphosate. The resulting finalplasmid was designated pMON66230 (FIG. 3).

A second AsnS2 construct was made using the aforementioned AsnS2(pMON79706) gene. The construct was made by insertion of the XbaI/EcoRIdigested AsnS2 gene into pMON61562, which had also been digested withXbaI and EcoRI, resulting in the AsnS2 gene being in operable linkagewith the NAS promoter and RGLUT1 terminator. The resulting plasmid wasdigested with NotI and ligated into the NotI digested pMON30167. Theresulting plasmid was designated pMON66229 (FIG. 2).

A third AsnS2 construct was made using the aforementioned AsnS2 gene(pMON79706). The P-FDA promoter used in this construct was isolated frompMON78810 by digestion with NotI and XbaI restriction enzymes. The P-FDApromoter was then ligated into pMON66246, which was previously digestedwith NotI and XbaI to remove its PPDK promoter. The resulting plasmidwas digested with NotI and ligated into the NotI digested pMON30167. Theresulting plasmid was designated pMON66231 (FIG. 4).

A fourth AsnS2 construct was made using the aforementioned AsnS2 gene(pMON79706). The P-RTBV promoter to be used in this construct wasgenerated by PCR from pMON74576. The 721 by fragment was digested withNotI and XbaI and ligated into pMON66246, which was previously digestedwith NotI and XbaI. The resulting plasmid, containing the AsnS2 gene inoperable linkage with the P-RTBV promoter and RGLUT1 terminator wasdigested with NotI and ligated into the NotI digested pMON30167. Theresulting plasmid was designated pMON66239 (FIG. 5).

A fifth AsnS2 construct was made using the aforementioned AsnS2 gene(pMON79706). A primer pair of ZmASsense,

-   -   5′TCCTAGACATGTCCGGCATACTTGCTG3′ (SEQ ID NO:46),        and ZmASantisense,    -   5′TGCAGAATTCTATCCCTCGATGG; (SEQ ID NO:47),        was used to amplify corn AsnS2 from pMON66240. PCR set up was as        follows: in a total volume of 50 ul PCR reaction, 1 μl of 10 mM        each primer of ZmASsense and ZmASantisense, 0.2 to 0.5 μg (1 μl)        of plasmid DNA of pMON66240, 5 μl of 10× AccuPrime™ Pfx Reaction        Mix, 1 μl of ACCuPrime™ Pfx DNA Polymerase (Invitrogen), and 41        μl of distilled water. The PCR reaction was carried out with the        following cycle parameters: 94° C. for 1 min., followed by 30        cycles of 94° C. for 15 seconds for denaturing; 58° C. for 15        sec of annealing, and 68° C. for 4 min.; followed by 10 min. of        extension at 68° C. The PCR product was purified using a PCR        purification kit from QIAGEN (QIAGEN Inc.). An aliquot of the        PCR corn AsnS2 product was digested with NcoI and EcoRI        restriction enzyme and another aliquot of the PCR product was        digested with AflIII and NcoI. The NcoI and EcoRI fragment was        then cloned into NcoI and EcoRI sites of pMON94901. The AflIII        and NcoI 5′ end fragment of corn AsnS2 was cloned into the NcoI        and EcoRI of the corn AsnS2 fragment at NcoI site. The resulting        plasmid (pMON74940), containing corn AsnS2 in operable linkage        with the e35S promoter and the Hsp17 terminator, was digested        with NotI and ligated into NotI digested pMON53616 to construct        pMON74946.

Each construct described above contained an expression cassette forexpression of a glyphosate insensitive Type II EPSPS as a means forselecting transgenic events (U.S. Pat. No. 5,633,435). The nucleic acidsequence of each construct was determined using standard methodology asset forth by PE Applied Biosystems BigDye terminator v.3.0 (PE AppliedBiosystems, Foster City, Calif.) and the integrity of the cloningjunctions confirmed. The pMON66229, pMON66230, pMON66231, pMON66239, andpMON74946 vectors were used in the subsequent transformation of corncells and regeneration of these cells into intact corn plants.Constructs of interest were introduced to immature embryos from cornline LH244 by an Agrobacterium-mediated transformation method, forinstance as described in U.S. Published Patent Application 20050048624.

Example 4 Protein and Amino Acid Analysis of Corn Seed Samples

This example sets forth a method of protein and amino acid analysis toselect seed of the present invention with increased asparagine andprotein using HPLC and near infrared measurements. For seed proteinanalysis, small bulk samples consisting of 50-100 seeds for eachtreatment were measured using near infrared transmittance spectroscopy(Infratec model 1221, Tecator, Hoganas Sweden). This procedure was basedupon the observation that a linear relation exists between theabsorption of near infrared radiation and the quantity of chemicalconstituents comprised in a typical seed sample. Prior to analyzingunknown samples, spectral data was collected with calibration samplesthat were subsequently analyzed using a primary analysis technique. Theprimary technique used was nitrogen combustion (Murray and Williams,1987). A multivariate model was developed using the spectral data fromthe spectrometer and the primary data. In the present case, a PLS-1(Partial Least Squares Regression Type I) multivariate model wasconstructed using 152 calibration samples. Each unknown sample wasscanned on the spectrometer at least five times and its protein contentpredicted with each scan. Each time the sample was scanned, it was addedback to the sample cuvette to provide an accurate representation of thesample tested. The predicted protein values were averaged for themultiple scans and then reported for each sample.

Free amino acid analysis was performed on corn tissues by HPLC. For eachsample, 20-50 mg lyophilized tissue were extracted with 1.5 mL of 10%trichloroacetic acid in 2-mL microfuge tubes. Samples were extracted atroom temperature overnight with gentle shaking. Extracted samples werecleared by centrifugation and the supernatant was removed for furtheranalysis. Free amino acid analysis was performed by HPLC on an AgilentSeries 1100 HPLC with a fluorescence detector and 96-well plateautosampler equipped with a Zorbax Eclipse AAA C18 column (4.6×75 mm,3.5 micron, Agilent Technologies, Palo Alto, Calif.) and Zorbax EclipseAAA analytical guard column (4.6×12.5 mm, 5 micron). Samples werepre-derivatized with o-pthalaldehyde immediately prior to separation.Free amino acids were resolved with a 40 mM phosphate buffer, pH7.6/Methanol/Acetonitrile gradient followed by fluorescence detection at340 nm/450 nm (excitation/emission). Free amino acids were quantifiedbased on external amino acid standards and peaks were integrated withChemStation software (Agilent). Relative standard deviations weretypically less than 8%.

Example 5 Field Evaluation of Asparagine Levels and Grain ProteinContent in Transgenic Corn Plants

This example sets forth the results of a field evaluation of the effectsof the corn AsnS constructs (pMON79706 and pMON92870) on asparagine andprotein levels in transformed corn plants and seed; and the effects ofthe corn AsnS constructs (pMON79700 and pMON79653) on grain proteincontent. The relative concentration of free asparagine in corn tissueswas obtained from inbred lines derived from R₀ corn plants transformedwith pMON79706 or pMON92870. For pMON79706, R₀ transformants werebackcrossed to the parent inbred, LH59, to create BC₁ seed. The BC₁seed, which segregates with the transgene, was planted in a fieldnursery and individual plants were scored for the presence of the NPTIImarker gene. Leaf tissue was collected for free amino acid analysis fromtransgene-positive and transgene-negative plants for each transgenicevent for free amino acid analysis. Leaf free amino acids of pMON79706transgenic plants were compared to negative isoline plants within eachevent and analyzed statistically by Student's T test with JMP 5.1software (SAS Institute, Cary, N.C.). For pMON92870, R₀ transformantswere backcrossed to the parent inbred, LH244, to create BC₁ seed. TheBC1 seed was planted in a field nursery and self-pollinated to createthe BC₁S₁ seed, which subsequently was planted in a second inbrednursery. Transgene-positive plants were identified for each transgenicevent following scoring for the presence of the NPTII marker gene. Leaftissue was collected from transgene-positive BC₁S₁ plants and parentalinbred plots planted at regular intervals in the nursery. Leaf freeamino acids for pMON92870 were analyzed statistically by performinganalysis of variance and comparing transgenic entries to the parentalcontrol by conducting Student's T test using SAS 9.1 software. For freeamino acid analyses for both constructs, leaf tissue was collected byremoval of an upper fully expanded leaf at anthesis followed by freezingon dry ice. Leaf samples were ground frozen, lyophilized, and measuredfor free amino acid content by HPLC.

Multiple transgenic events of pMON79706 and pMON92870 were observed toshow substantial increases in leaf asparagine content (Table 2). Four ofseven events of pMON79706 tested showed significant increases in theconcentration of leaf asparagine, as indicated by a p value of 0.05 orless. In transgenic events of pMON92870, expressing a second maizeasparagine synthetase gene, four of five events showed significantincreases in leaf asparagine levels (Table 2). These data show thattransgenic expression of maize AsnS2 and maize AsnS3 under the riceactin promoter in pMON79706 and pMON92870, respectively, can result in aspecific increase in free asparagine, which is consistent with theoverexpression of active asparagine synthetase.

The relative concentration of protein in corn seed was obtained frominbred lines derived from R₀ corn plants transformed with pMON79706 orpMON92870. BC₁ transgenic plants of pMON79706 (described above) wereself-pollinated and the resulting BC₁S₁ grain was grown to maturity andmeasured for protein content by single ears. Protein was measured as apercentage of dry weight at 0% moisture. Grain protein for pMON79706transgenic plants were compared to negative isoline plants within eachevent and analyzed statistically by Student's T test with SAS 9.1software. For pMON98270, BC₁S₁ plants were self-pollinated and grown tomaturity and measured for protein content by single ears. Grain proteinfor pMON92870 was analyzed statistically with a custom developed spatialmethod by conducting a by-location analysis. The by-location analysis isa two-step process. The first step in the analysis involved estimatingthe spatial autocorrelation in the field by fitting an anisotropicspherical semi-variogram model using all spatial check plots that wereplaced systematically in the field (every 6th plot). The second stage ofanalysis involved adjusting the values of the transgenic entries for thespatial variability using the spatial autocorrelation structureestimated in the first stage of the analysis. Following the adjustmentfor spatial autocorrelation, mean comparison was carried out where themean value of a transgenic entry was compared to the parental control totest the statistical significance of the difference between a transgeneand the control mean.

Multiple events of both pMON79706 and pMON92870 showed significantincreases in inbred grain protein content (Table 3). Three of fiveevents of pMON79706 that were analyzed statistically showed significantincreases in grain protein content (p<0.05) and two other events showedtrends toward significant increases (p<0.15). Two events did not returnsufficient numbers of ears for a statistical analysis. Three of fourtransgenic events of pMON92870 showed significant increases in grainprotein content (p<0.1), with one event untested due to insufficientnumbers of ears for analysis. These data confirm that pMON79706 andpMON92870 produce transgenic events that increase grain protein contentin maize in addition to increasing leaf asparagine content.

TABLE 2 Relative leaf asparagine concentrations in inbred maizetransformed with corn AsnS2 gene (pMON79706) or corn AsnS3 gene(pMON92870). Mean of Mean of Transgene- Transgene- p Construct^(a) EventGeneration positive Plants^(b) negative Plants Diffe ence valuepMON79706 ZM_M50965 BC₁ 16.3 10.7 5.6 0.319 ZM_M50973 BC₁ 32.0 7.3 24.30.025 ZM_M50974 BC₁ 25.0 5.3 19.8 0.014 ZM_M50980 BC₁ 18.0 5.3 12.50.001 ZM_M50984 BC₁ 29.3 10.3 19.1 0.002 ZM_M50985 BC₁ 15.7 6.3 9.50.278 ZM_M51011 BC₁ 15.0 7.3 7.7 0.191 pMON92870 ZM_M102252 BC₁S₁ 22.50.0 22.5 <0.001 ZM_M103304 BC₁S₁ 18.8 0.0 18.8 <0.001 ZM_M103315 BC₁S₁30.6 0.0 30.6 <0.001 ZM_M103316 BC₁S₁ 2.6 0.0 2.6 0.55 ZM_M103320 BC₁S₁30.0 0.0 30.0 <0.001 ^(a)Leaf asparagine was determined in two separateexperiments for pMON79706 and pMON92870. ^(b)Relative free asparaginemeasured as a percentage of total free amino acids in leaf tissue

TABLE 3 Grain protein content in inbred maize transformed with maizeAsnS2 gene (pMON79706) or maize AsnS3 gene (pMON92870). Mean of Mean ofTransgene- Transgene- p Construct^(a) Event Generation positivePlants^(a) negative Plants Difference value pMON79706 ZM_M50965 BC₁ nd^(c) nd nd nd ZM_M50973 BC₁ 15.1 11.6 3.5 0.024 ZM_M50974 BC₁ nd ndnd nd ZM_M50980 BC₁ 13.8 12.0 1.8 0.118 ZM_M50984 BC₁ 15.1 11.4 3.70.002 ZM_M50985 BC₁ 13.9 10.8 3.1 0.003 ZM_M51011 BC₁ 13.5 11.4 2.2 0.08pMON92870 ZM_M102252 BC₁S₁ 13.3 11.9 1.4 0.096 ZM_M103304 BC₁S₁ 13.711.9 1.8 0.042 ZM_M103315 BC₁S₁ nd 11.9 nd nd ZM_M103316 BC₁S₁ 11.2 11.9−0.7 0.373 ZM_M103320 BC₁S₁ 14.2 11.9 2.3 0.003 ^(a)Grain protein wasdetermined in two separate experiments for pMON79706 and pMON92870.^(b)Grain protein measured as a percentage of total grain composition ona 0% moisture basis. ^(c)nd; not determined.

The high asparagine and grain protein phenotype pMON79706 was confirmedin multiple tissues in a second trial. After the BC₁ generation, fiveevents of pMON79706 were self-pollinated in two following nurseries togenerate BC₁S₃ seed that was homozygous for the transgene. The relativeconcentration of asparagine resulting from expression of the pMON79706construct was determined in a study at the corn V8 growth stage bycomparing homozygous BC₁S₃ plants and a LH59 corn variety control (Table4). Transgenic entries and controls were planted in a randomizedcomplete block design with 5 replicated blocks in a field plot. Theupper fully expanded leaves and stem sections of two plants were sampledand pooled, placed on dry ice, ground, lyophilized, and measured forfree amino acid content by HPLC. Values followed by “*” indicate asignificant difference from the LH59 control (Dunnett's one-tail test;(SAS 9.1, Cary, N.C.). Asparagine measurements taken at both the V8growth stage and the R₁ generation showed that plants from fivepMON79706 events had significant increases in free asparagine. Relativefree asparagine levels in V8 leaf tissue were increased up to 13.9% ascompared to 3.4% in the LH59 variety control, and stem asparagine wasincreased up to 39% as compared to 9.6 in the control (Table 4). Forgrain protein analysis, 10 ears were sampled per plot, shelled, andanalyzed for grain protein concentration. Grain protein was alsoincreased significantly in the five events of pMON79706 (Table 4). Theresults show that, as a general trend, events producing a significantincrease in asparagine also produced as significant increase in kernelprotein (Tables 2-4).

TABLE 4 Relative asparagine concentrations at V8 growth stage and grainprotein concentration at maturity in BC₁S₃ corn plants transformed withthe corn AsnS2 gene (pMON79706). Leaf Stem Grain Asn % Asn (ppm) Asn %Asn (ppm) Protein % Event Mean Mean Mean Mean Mean LH59 control 3.54 3899.6 2254 12.3 ZM_M50974 12.31* 1312* 32.20*  9179* 14.8* ZM_M5098010.20* 1058* 38.68* 12844* 15.2* ZM_M50984 9.18*  997* 28.20*  8062*14.4* ZM_M50985 5.86* 697 15.12* 3404 14.5* ZM_M51011 13.89* 1740*37.05* 11820* 15.0* *Significant at p < 0.05

Significant increases in hybrid grain protein were observed for threedifferent constructs expressing asparagine synthetase genes under therice actin promoter. Homozygous inbred corn lines were produced from R₀transgenic events of pMON79706 (corn AsnS2), pMON79700 (soy AsnS), andpMON79653 (yeast AsnS1) by first backcrossing R₀ events to the recurrentparent, LH59, followed by self-pollinations of transgene-positiveselections in two subsequent inbred nurseries using the NPTII selectablemarker to score for zygosity. The homozygous events for each constructwere then used as a male pollen donor in a cross with a female inbredline to create the F₁ hybrid. The F₁ hybrid seed was planted in amultiple-location trial and transgenic events for each construct wereanalyzed for final grain protein and compared to the recurrent parenthybrid control following a spatial correction analysis based on grainprotein in control hybrids that were planted at regular intervalsthroughout the field. Grain was harvested from each plot, shelled, andanalyzed for protein content. Data were analyzed using a customdeveloped spatial method by conducting a by-location and an acrosslocation analysis. The by-location analysis is a two-step process. Thefirst step in the analysis involved estimating the spatialautocorrelation in the field by fitting an anisotropic sphericalsemi-variogram model using all spatial check plots that were placedsystematically in the field (every 3rd plot). The second stage ofanalysis involved adjusting the values of the transgenic entries for thespatial variability using the spatial autocorrelation structureestimated in the first stage of the analysis. Following the adjustmentfor spatial autocorrelation in each location separately, anacross-location analysis was conducted where the mean value of atransgenic entry was compared to the parental control to test thestatistical significance (P=0.20) of the difference between a transgeneand the control mean. All five events of pMON79706 showed significantincreases in grain protein in the hybrid trial, consistent with theobservation that grain protein was increased in the inbred lines oftransgenic events of this construct (Table 5). Two other asparaginesynthetase constructs, pMON79700 (soy AsnS) and pMON79653 (yeast AsnS),also showed significant increases in grain protein levels in two of fiveevents and two of two events, respectively.

TABLE 5 Grain protein content in hybrid maize transformed with genes forasparagine synthetase from maize (Zea mays), soy (Glycine max), andyeast (Saccharomyces cerevisiae)^(a). Protein Trans- Protein Pro- genicControl tein p Construct Gene Event Mean Mean Delta value pMON79706Maize ZM_M50974 11.12 8.65 2.48 0.000 AsnS2 ZM_M50980 9.17 8.65 0.530.003 ZM_M50984 9.56 8.65 0.91 0.000 ZM_M50985 9.71 8.65 1.07 0.000ZM_M51011 9.45 8.65 0.81 0.000 pMON79700 Soy ZM_M49436 8.52 8.65 −0.130.469 AsnS ZM_M61615 11.25 8.65 2.61 0.000 ZM_M62422 13.30 8.65 4.650.000 ZM_M62428 8.61 8.65 −0.04 0.826 ZM_M64520 8.76 8.65 0.11 0.570pMON79653 Yeast ZM_M49883 9.12 8.65 0.48 0.007 AsnS1 ZM_M65281 9.43 8.650.79 0.000 ^(a)Grain protein measured as a percentage of total graincomposition on a 0% moisture basis.

Example 6 Field Evaluation of the Transgene Expression and AsparagineSynthetase Enzyme Activity Due to pMON79706 and pMON92870

Transgene expression was confirmed in transgenic events of pMON79706 andpMON92870. For pMON79706, tissue used for the determination of leafasparagine content in the field trial with BC₁S₃ homozygous inbred lineswas also used for determination of transgene expression based onmeasurement of the expression from the 3′-terminator sequence (St.Pis4)from pMON79706 at anthesis. Two leaf samples were harvested and pooledfrom each of 5 replicate plots (10 for inbred control) and frozen on dryice. Leaf samples were then ground frozen for expression analysis. ForRNA extraction, 50 mg of frozen tissue were aliquoted into 96-wellplates. Each sample was extracted with 500 μl of lysis buffer containinga 1:1 solution of ABI nucleic acid lysis solution (Applied Biosystems,Foster City, Calif.) to 1×PBS ph7.4 (without MgCl or CaCl). RNA wasextracted from fresh-frozen tissue samples using filter-plates tocapture nucleic acids from crude lysates, and 50 μl of ABI elutionbuffer was used to elute bound RNA. Quantitative PCR was performed usinga 5 μl RNA template with 5 μl ABI one-step RT-PCR reagent. The reactionswere carried out for 40 PCR cycles on an ABI Taqman 7900 PCR instrument,with cycling parameters of 48° C. for 30 min., 95° C. for 10 min., 95°C. for 10 sec., 60° C. for 1 min. Fluorescent measurements were takenfrom each well at each of the 40 cycles for both the terminator sequencederived from the potato protease inhibitor II (St.Pis4) and theendogenous control (ubiquitin). A subset of samples was run withoutreverse transcriptase to monitor DNA contamination. Samples were scoredfor relative expression by subtracting the cycle threshold values forSt.Pis4 from the cycle threshold value of the endogenous control. Thecycle threshold (Ct) was determined, and the delta Ct was calculatedfrom the St.Pis4 minus endogenous control value. An in situ wild-typewas created by calculating the average endogenous control signals andsetting the St.Pis4 signal value at 40. The delta Ct of the unknownsamples was subtracted from the delta Ct of the in situ wild-type. Finaldata was reported as pinII (St.Pis4) expression relative to wild type.Quantitative RT-PCR analysis confirmed overexpression of the transgenefrom six of six events of pMON79706 (FIG. 6).

Transgene expression was also confirmed in inbred events comprisingpMON92870. RNA expression was determined from leaf tissue at anthesis ofinbred plants grown in a field nursery by first harvesting an upperexpanded leaf from each plant (4-8 plants per event) and freezing on dryice. Transgene-positive plants were previously identified based onpresence of the NPTII marker gene. Leaf tissue was ground while frozen,and analyzed for expression from the 3′-terminator sequence (St.Pis4) ofpMON92870. Quantitative RT-PCR analysis showed that five of six eventscomprising pMON92870 showed increased transgene expression as comparedto an inbred control (FIG. 7). The low RNA expression in pMON92870 eventZM_M103316 is consistent with the low leaf asparagine content and grainprotein content in this event.

The effect of expression of asparagine synthetase genes on asparaginesynthetase activity was measured in transgenic events of pMON79706 andpMON92870. Frozen, ground leaf tissue was aliquoted (200-400 mg) intowells from a precooled 96 deep-well plate. Protein was extracted inBuffer A (100 mM Hepes-OH, pH 8.0, 0.1 mM EDTA, 10 mM MgCl₂, 2 mMaspartate, 0.5 mM DTT, 67 mM mercaptoethanol, 20% (v/v) glycerol, 0.1 mMATP, 1% (v/v) P9599 (Sigma Company), 25 mM KCl). A small amount of sandwas added to each well. Buffer A was then added to the leaf tissue inthe wells at a ratio of 4:1 (buffer:tissue). The plates were thenagitated in a paint shaker for 2 min. to mix the sample and thencentrifuged at 5000×g for 10 minutes. The supernatant (100-200 μL) wasdesalted in a 96-well macro spin plate (SNS S025L, The Nest Group Inc.,Southboro, Mass.) equilibrated in buffer A. The supernatant was theneither assayed immediately or frozen in liquid nitrogen and maintainedat −80° C. until used. To assay asparagine synthetase activity, desaltedprotein extracts (10-50 μL) were added to wells containing 100 μL assaysolution (100 mM Hepes, pH 8.0, 10 mM MgCl₂, 2 mM aspartate, 5 mM DTT,10 mM ATP, 1 mM amino(oxy)acetic acid (aspartate amino transferaseinhibitor), 1 mM aspartic semialdehyde (asparaginase inhibitor). Tostart the reaction, glutamine (final concentration of 2 mM for standardassay) was added to the solution, which was then mixed. The assaymixture was then incubated for 1 to 2 hours. The reaction was thenstopped by the addition of an equal volume of 20% (w/v) trichloroaceticacid. The mixture was then filtered to remove precipitate and asparaginewas measured by HPLC. Sample size was increased from 0.5 μL to 2.5 μLfor HPLC, excitation wavelength was reduced from 340 nm to 235 nm, andfluorimeter gain was increased from 10 to 13. This results in asensitivity of detection of 0.5 to 100 μM asparagine and allows themeasurement of levels of activity in the 100s of microunits.

For pMON79706, tissue used for the determination of leaf asparaginesynthetase enzyme activity was from a field trial with BC₁S₃ homozygousinbred lines harvested at the V7 growth stage. Events of pMON79706 wereshown to display increased leaf asparagine synthetase activity (Table6). Asparagine synthetase activity was increased up to 5-fold over theinbred variety control. Asparagine synthetase enzyme activity was alsodetermined for transgenic events of pMON92870 in an inbred field nurseryat the time of anthesis. Four of five pMON92870 events also showedincreased enzyme activity (Table 6). The increased asparagine synthetaseenzyme activity in corn plants expressing the corn AsnS2 (pMON79706) orcorn AsnS3 (pMON92870) under the rice actin promoter is consistent withthe increase in gene expression and leaf asparagine increases observedwith these constructs.

TABLE 6 Asparagine synthetase activity in inbred lines of transgenicevents of pMON79706 and pMON92870^(a). Construct Event AsnS Activity(μunits/mg protein) Control LH59 276 pMON79706 ZM_M50973 519 ZM_M509741179 ZM_M50984 1592 ZM_M50985 450 ZM_M51011 1031 Control LH244 98pMON92870 ZM_M102252 160 ZM_M103304 209 ZM_M103315 243 ZM_M103316 11ZM_M103319 192 ZM_M103320 240 ^(a)Enzyme activities for pMON79706 andpMON92870 were determined from two different field experiments.

Example 7 Field Evaluation of the Effects of pMON66231, pMON66239, andpMON74946 on Asparagine and Grain Protein Content

The relative content of free asparagine in corn tissues was obtainedfrom hybrid lines derived from R₀ corn plants (LH244 background)transformed with pMON66231 (FIG. 4), where corn AsnS2 is under thecontrol of the corn FDA promoter. Hybrids were made by crossing the R₀plants to the male inbred line LH59, which creates a segregating (1:1)F₁ population. The resulting F₁ seed was planted in three midwestlocation with two replications at each location. Plots were sprayed withglyphosate at V3 growth stage to eliminate null segregants. A hybridcontrol was planted in the perimeter and comparisons were made to thehybrid control. Upper leaves were collected and pooled from three plantswithin each plot at the time of anthesis, two hours after sunset, at allthree locations. Leaves were placed immediately on dry ice and thenstored at −80° C. until processing. Leaves were ground frozen, and aportion was lyophilized for free amino acid analysis by HPLC. Data werefirst screened for outliers with the two-pass method for deletedstudentized residuals using Bonferroni-adjusted p-values. Outliers wereidentified and removed from the data set before analysis of variancecalculations were initiated. The data were analyzed according to anacross-locations randomized complete block design. Construct-eventcombinations were modeled with fixed effects, and locations and repswithin locations were modeled with random effects. Treatment comparisonswere made by performing contrasts of the least-squares means of theconstruct-event combinations. Relative leaf asparagine was increasedsignificantly in 11 of 12 events of pMON66231, with asparagine levels ashigh as 16% as compared to 3% in the control (Table 7). Mature grainprotein was also measured following harvest of 10 ears per plot followedby shelling and pooling of seed for each plot, which was then measuredfor grain protein content. Nine of 12 events were found to significantlyincrease protein content in the mature grain over the LH244/LH59 hybridcontrol.

TABLE 7 Relative leaf asparagine and mature grain protein content inpMON66231 transgenic events. Leaf Asn %^(a) Grain Protein % Event Mean pvalue^(b) Mean p value^(b) LH244/LH59 2.73 8.68 ZM_S120303 11.41 <.0018.57 0.774 ZM_S120316 8.92 0.007 9.90 0.002 ZM_S122246 8.69 0.01 10.22<.001 ZM_S122249 9.85 0.002 10.83 <.001 ZM_S122257 9.57 0.003 12.48<.001 ZM_S122262 10.10 0.001 9.70 0.011 ZM_S122267 9.33 0.004 9.23 0.162ZM_S122279 12.67 <.001 11.13 <.001 ZM_S122280 12.54 <.001 10.90 <.001ZM_S122281 9.47 0.003 10.53 <.001 ZM_S122291 16.25 <.001 9.83 0.004ZM_S122303 6.44 0.126 8.53 0.71 ^(a)Relative free asparagine measured asa percentage of total free amino acids in leaf tissue ^(b)Compared tohybrid control.

The relative content of free asparagine in corn tissues was obtainedfrom hybrid lines derived from R₀ corn plants (LH244 background)transformed with pMON66239 and pMON74946, where corn AsnS2 is under thecontrol of the RTBV or e35S promoter, respectively. Hybrids were made bycrossing the R₀ plants to the male inbred line, LH59, which creates asegregating (1:1) F₁ population. The resulting F₁ seed was planted inone location in Hawaii with three replications for each transgenicevent. Plots were sprayed with glyphosate at V3 growth stage toeliminate null segregants. A hybrid control lacking the corn AsnS2 genewas included for comparison. Upper leaves were collected and pooled fromthree plants within each plot at the time of anthesis, two hours aftersunset. Leaves were placed immediately on dry ice and then stored at−80° C. until processing. Leaves were ground frozen, and a portion waslyophilized for free amino acid analysis by HPLC. Data were firstscreened for outliers with the two-pass method for deleted studentizedresiduals using Bonferroni-adjusted p-values. Outliers were identifiedand removed from the data set before analysis of variance calculationswere initiated. The data were analyzed according to a randomizedcomplete block design. Construct-event combinations were modeled withfixed effects, and reps were modeled with random effects. Treatmentcomparisons were made by performing contrasts of the least-squares meansof the construct-event combinations. Relative leaf asparagine wasincreased significantly in 10 of 13 events of pMON74946, with asparaginelevels as high as 16% as compared to 2% in the control (Table 8). Maturegrain protein was also measured following harvest of all ears per plotfollowed by shelling and pooling of seed for each plot, which was thenmeasured for grain protein content and analyzed statistically as for theleaf asparagine trait. Ten of thirteen events were found to possesssignificantly increased protein content in the mature grain as comparedto the hybrid control, and the same 10 events with increased leafasparagine also showed increased protein in the hybrid trial. Fortransgenic events of pMON66239, 11 of 15 events showed increases in leafasparagine content, and 3 of 15 events showed significant increases ingrain protein at the 0.05 alpha level, although an additional fivetransgenic events showed increased protein at p<0.15, indicating thatexpression of corn AsnS2 under the RTBV promoter (pMON66239) canincrease leaf asparagine content and kernel protein content, but to alesser extent than under the e35s promoter (pMON74946) (Table 8).

TABLE 8 Relative leaf asparagine and mature grain protein content inpMON74946 and pMON66239 transgenic events. Leaf Asn %^(a) Grain Protein% Construct Event Mean p value^(b) Mean p value^(b) Control Hybridcontrol 1.47 7.98 pMON74946 ZM_S156600 10.37 <.0001 8.67 0.0398ZM_S156602 1.23 0.7315 8.43 0.1728 ZM_S156606 0.39 0.1214 7.43 0.0995ZM_S156613 0.71 0.2786 7.70 0.3959 ZM_S156634 14.79 <.0001 9.37 <.0001ZM_S156636 12.28 <.0001 9.23 0.0002 ZM_S160005 15.57 <.0001 9.50 <.0001ZM_S160015 15.85 <.0001 13.10 <.0001 ZM_S160025 13.41 <.0001 9.13 0.0007ZM_S160026 11.94 <.0001 9.17 0.0005 ZM_S160034 11.00 <.0001 9.60 <.0001ZM_S160037 15.79 <.0001 9.10 0.001 ZM_S160042 14.73 <.0001 9.17 0.0005pMON66239 ZM_S140597 8.84 <.0001 11.03 <.0001 ZM_S140601 2.14 0.34198.20 0.5078 ZM_S140609 10.08 <.0001 8.50 0.1182 ZM_S140613 2.67 0.08818.50 0.1182 ZM_S140615 1.23 0.7333 8.20 0.5078 ZM_S140617 6.68 <.00018.50 0.1182 ZM_S140618 3.93 0.0005 8.73 0.0244 ZM_S140633 5.96 <.00018.63 0.0503 ZM_S140635 3.69 0.0017 8.37 0.2445 ZM_S140645 5.88 <.00018.37 0.2445 ZM_S140647 3.66 0.0019 8.30 0.3353 ZM_S140651 4.66 <.00018.57 0.0784 ZM_S140661 4.13 0.0002 8.03 0.8741 ZM_S140663 2.07 0.61 9.030.0018 ZM_S140665 7.33 <.0001 8.27 0.388 ^(a)Relative free asparaginemeasured as a percentage of total free amino acids in leaf tissue^(b)Compared to hybrid control.

All publications, patents and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

The function of polypeptides of the present invention is determined bycomparison of the amino acid sequence of the novel polypeptides to aminoacid sequences of known polypeptides. A variety of homology based searchalgorithms are available to compare a query sequence to a proteindatabase, including for example, BLAST, FASTA, and Smith-Waterman. Inthe present application, BLASTX and BLASTP algorithms are used toprovide protein function information. A number of values are examined inorder to assess the confidence of the function assignment. Usefulmeasurements include “E-value” (also shown as “hit_p”), “percentidentity”, “percent query coverage”, and “percent hit coverage”.

In BLAST, E-value, or expectation value, represents the number ofdifferent alignments with scores equivalent to or better than the rawalignment score, S, that are expected to occur in a database search bychance. The lower the E value, the more significant the match. Becausedatabase size is an element in E-value calculations, E-values obtainedby BLASTing against public databases, such as GenBank, have generallyincreased over time for any given query/entry match. In setting criteriafor confidence of polypeptide function prediction, a “high” BLAST matchis considered herein as having an E-value for the top BLAST hit providedin Table 9 of less than 1E-30; a medium BLASTX E-value is 1E-30 to 1E-8;and a low BLASTX E-value is greater than 1E-8. The top BLAST hit andcorresponding E values are provided in columns six and seven of Table 9.

Percent identity refers to the percentage of identically matched aminoacid residues that exist along the length of that portion of thesequences which is aligned by the BLAST algorithm. In setting criteriafor confidence of polypeptide function prediction, a “high” BLAST matchis considered herein as having percent identity for the top BLAST hitprovided in Table 9 of at least 70%; a medium percent identity value is35% to 70%; and a low percent identity is less than 35%.

Of particular interest in protein function assignment in the presentinvention is the use of combinations of E-values, percent identity,query coverage and hit coverage. Query coverage refers to the percent ofthe query sequence that is represented in the BLAST alignment. Hitcoverage refers to the percent of the database entry that is representedin the BLAST alignment. In the present invention, function of a querypolypeptide is inferred from function of a protein homolog where either(1) hit_p<1e-30 or % identity>35% AND query_coverage>50% ANDhit_coverage>50%, or (2) hit_p<1e-8 AND query_coverage>70% ANDhit_coverage>70%.

The open reading frame in each polynucleotide sequence is identified bya combination of predictive and homology based methods. The longest openreading frame (ORF) is determined, and the top BLAST match is identifiedby BLASTX against NCBI. The top BLAST hit is then compared to thepredicted ORF, with the BLAST hit given precedence in the case ofdiscrepancies.

Functions of polypeptides encoded by the polynucleotide sequences of thepresent invention are determined using a hierarchical classificationtool, termed FunCAT, for Functional Categories Annotation Tool. Mostcategories collected in FunCAT are classified by function, to althoughother criteria are used, for example, cellular localization or temporalprocess. The assignment of a functional category to a query sequence isbased on BLASTX sequence search results, which compare two proteinsequences. FunCAT assigns categories by iteratively scanning through allblast hits, starting with the most significant match, and reporting thefirst category assignment for each FunCAT source classification scheme.In the present invention, function of a query polypeptide is inferredfrom the function of a protein homolog where either (1) hit_p<1e-30 or %identity >35% AND query_coverage >50% AND hit_coverage >50%, or (2)hit_p<1e-8 AND query_coverage >70% AND hit_coverage>70%.

Functional assignments from five public classification schemes, GO_BP,GO_CC, GO_MF, KEGG, and EC, and one internal Monsanto classificationscheme, POT, are provided in Table 9. The column under the heading“CAT_TYPE” indicates the source of the classification. GO_BP=GeneOntology Consortium—biological process; GO_CC=Gene OntologyConsortium—cellular component; GO_MF=Gene Ontology Consortium—molecularfunction; KEGG=KEGG functional hierarchy; EC=Enzyme Classification fromENZYME data bank release 25.0; POI=Pathways of Interest. The columnunder the heading “CAT_DESC” provides the name of the subcategory intowhich the query sequence was classified. The column under the heading“PRODUCT_HIT_DESC” provides a description of the BLAST hit to the querysequences that led to the specific classification. The column under theheading “HIT_E” provides the e-value for the BLAST hit. It is noted thatthe c-value in the HIT_E column may differ from the e-value based on thetop BLAST hit provided in the E-VALUE column since these calculationswere done on different days, and database size is an element in E-valuecalculations. E-values obtained by BLASTing against public databases,such as GenBank, will generally increase over time for any givenquery/entry match.

Table 9 Column Descriptions:

SEQ_NUM provides the SEQ ID NO for the listed polynucleotide sequences.

CONTIG_ID provides an arbitrary sequence name taken from the name of theclone from which the cDNA sequence was obtained.

PROTEIN_NUM provides the SEQ ID NO for the translated polypeptidesequence

NCBI_GI provides the GenBank ID number for the top BLAST hit for thesequence. The top BLAST hit is indicated by the National Center forBiotechnology Information GenBank Identifier number.

NCBI_GI_DESCRIPTION refers to the description of the GenBank top BLASThit for the sequence.

E_VALUE provides the expectation value for the top BLAST match.

MATCH_LENGTH provides the length of the sequence which is aligned in thetop BLAST match

TOP_HIT_PCT_IDENT refers to the percentage of identically matchednucleotides (or residues) that exist along the length of that portion ofthe sequences which is aligned in the top BLAST match.

CAT_TYPE indicates the classification scheme used to classify thesequence. GO_BP=Gene Ontology Consortium—biological process; GO_CC=GeneOntology Consortium—cellular cellular component; GO_MF=Gene OntologyConsortium—molecular function; KEGG=KEGG functional hierarchy(KEGG=Kyoto Encyclopedia of Genes and Genomes); EC=Enzyme Classificationfrom ENZYME data bank release 25.0; POI=Pathways of Interest.

CAT_DESC provides the classification scheme subcategory to which thequery sequence was assigned.

PRODUCT_CAT_DESC provides the FunCAT annotation category to which thequery sequence was assigned.

PRODUCT_HIT_DESC provides the description of the BLAST hit whichresulted in assignment of the sequence to the function category providedin the cat_desc column.

HIT_E provides the E value for the BLAST hit in the hit_desc column.

PCT_IDENT refers to the percentage of identically matched nucleotides(or residues) that exist along the length of that portion of thesequences which is aligned in the BLAST match provided in hit_desc.

QRY_RANGE lists the range of the query sequence aligned with the hit.

HIT_RANGE lists the range of the hit sequence aligned with the query.

QRY_CVRG provides the percent of query sequence length that matches tothe hit (NCBI) sequence in the BLAST match (% qry cvrg=(matchlength/query total length)×100).

HIT_CVRG provides the percent of hit sequence length that matches to thequery sequence in the match generated using BLAST (% hit cvrg=(matchlength/hit total length)×100).

TABLE 9 Seq_Num Seq_ID PROTEIN_NUM CDNA_Coord Prt_Num Blast_Hit_ID 1.13515 JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 2. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 3. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 4. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 5. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 6. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 7. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08__FLI.pep 8. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 9. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08FLI AA08_FLI.pep 10. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08__FLI AA08_FLI.pep 11. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 12. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 13. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 14. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 15. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 16. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 17. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08FLI.pep 18. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 19. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08FLI.pep 20. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 21. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 22. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 23. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 24. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 25. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 26. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 27. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 28. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 29. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 30. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 31. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 32. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 33. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 34. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 35. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 36. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 37. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 38. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 39. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 40. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 41. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_,FLI.pep 42. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 43. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 44. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 45. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 46. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 47. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 48. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 49. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 50. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 51. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 52. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 53. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep 54. 13515JC-ZMXLIB3526P040 JC-ZMXLIB3526P040 3-1811gi|1351989|sp|P49094|ASNS_MAIZE AA08_FLI AA08_FLI.pep

Blast_EValue Blast_FR_ID Blast_Des Species Cat_Type Cat_Desc 1. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI 5.1.3.4.16.7.17.87hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 2. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.21.7.17.97 hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 3. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.7.17.97 hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 4. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.11.13.4.15.7.17.97 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 5. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.11.13.4.16.7.17.97 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 6. 0.00 0.79Asparagine synthetase [glutamine- Zea mays POI 5.1.11.13.4.7.17.97hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 7. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.2.3.11.13.4.15.7.17.97 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 8. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.2.3.11.13.4.16.7.17.97 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 9. 0.00 0.79Asparagine synthetase [glutamine- Zea mays POI 5.1.2.3.11.13.4.7.17.97hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 10. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.2.3.4.16.7.17.97 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 11. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.2.4.15.7.17.97 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 12. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI 5.1.7.17.87hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 13. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.2.4.7.17.97 hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 14. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.4.7.17.87 hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 15. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.3.11.13.4.15.7.17.97 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 16. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.3.11.13.4.16.7.17.97 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 17. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI5.1.3.11.13.4.7.17.97 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 18. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.3.4.15.7.17.97 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 19. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI 5.1.3.4.16.7.17.97hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 20. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.3.4.7.17.97 hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 21. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.4.15.7.17.97 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 22. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.4.16.7.17.97 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 23. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI 5.1.4.7.17.97hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 24. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.2.4.16.7.17.97 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 25. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.2.4.15.7.17.87 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 26. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI 5.1.4.15.7.17.87hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 27. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays EC6.3.5.4 hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 28. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.11.13.4.15.7.17.87 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 29. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.11.13.4.16.7.17.87 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 30. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI 5.1.11.13.4.7.17.87hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 31. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.2.3.11.13.4.15.7.17.87 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 32. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.2.3.11.13.4.16.7.17.87 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 33. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI5.1.2.3.11.13.4.7.17.87 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 34. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.2.3.4.15.7.17.87 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 35. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI 3.7.110.579hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 36. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.2.3.4.7.17.87 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 37. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.2.3.4.7.17.97 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 38. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI 5.1.2.4.16.7.17.87hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 39. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.2.4.7.17.87 hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 40. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.21.7.17.87 hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 41. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.3.11.13.4.15.7.17.87 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 42. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.3.11.13.4.16.7.17.87 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 43. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI5.1.3.11.13.4.7.17.87 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 44. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.3.4.15.7.17.87 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 45. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI 5.1.3.4.7.17.87hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 46. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays POI5.1.4.16.7.17.87 hydrolyzing] (Glutamine-dependent asparaginesynthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 47. 0.00 0.79 Asparagine synthetase[glutamine- Zea mays POI 5.1.2.3.4.16.7.17.87 hydrolyzing](Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 48. 0.000.79 Asparagine synthetase [glutamine- Zea mays POI 5.1.2.3.4.15.7.17.97hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 49. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays KEGG 2.5hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 50. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays MIPS EC3.2.1.3 hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 51. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays KEGG 7.1hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 52. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays MIPS_EC6.3.5.4 hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 53. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays KEGG 5.2hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 54. 0.00 0.79 Asparagine synthetase [glutamine- Zea mays EC3.2.1.3 hydrolyzing] (Glutamine-dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays]

Prod_Cat_Desc 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Ligases\Forming carbon-nitrogenbonds\Carbon--nitrogen ligases with glutamine asamido-N-donor\Asparagine synthase (glutamine-hydrolysing) 28. 29. 30.31. 32. 33. 34. 35. Monsanto Patent Pathways\Yield\Glutamate andGlutamine Family Synthesis/Degradation\Asparagine synthase 36. 37. 38.39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. Energy Metabolism\Nitrogenmetabolism [MAP:00910] 50. Hydrolases. 3.-.-.- Hydrolases.\Glycosidases.3.2.-.- Glycosidases.\ Hydrolysing O-glycosyl compounds. 3.2.1.-Hydrolysing O-glycosyl compounds.\Glucan 1,4-alpha-glucosidase.3.2.1.3Glucan 1,4-alpha-glucosidase. 51. Metabolism of ComplexCarbohydrates\Starch and sucrose metabolism [MAP:00500] 52. Ligases.6.-.-.- Ligases.\Forming carbon-nitrogen bonds. 6.3.-.- Formingcarbon-nitrogen bonds.\Carbon--nitrogen ligases with glutamine asamido-N-donor. 6.3.5.- Carbon--nitrogen ligases with glutamine asamido-N-donor.\Asparagine synthase (glutamine- hydrolysing).6.3.5.4Asparagine synthase (glutamine-hydrolysing). 53. Amino AcidMetabolism\Alanine and aspartate metabolism [MAP:00252] 54.Hydrolases\Glycosylases\Glycosidases - hydrolysing O-glycosylcompounds\Glucan 1,4-alpha-glucosidase

FunCat_S_Beg FunCat_S_End FunCat_Strand FunCat_E-Value FunCat_Hit_ID 1.1.00 586.00 + 0.00 g1351989 2. 1.00 544.00 + 0.00 g3821280 3. 1.00544.00 + 0.00 g3821280 4. 1.00 544.00 + 0.00 g3821280 5. 1.00 544.00 +0.00 g3821280 6. 1.00 544.00 + 0.00 g3821280 7. 1.00 544.00 + 0.00g3821280 8. 1.00 544.00 + 0.00 g3821280 9. 1.00 544.00 + 0.00 g382128010. 1.00 544.00 + 0.00 g3821280 11. 1.00 544.00 + 0.00 g3821280 12. 1.00586.00 + 0.00 g1351989 13. 1.00 544.00 + 0.00 g3821280 14. 1.00 586.00 +0.00 g1351989 15. 1.00 544.00 + 0.00 g3821280 16. 1.00 544.00 + 0.00g3821280 17. 1.00 544.00 + 0.00 g3821280 18. 1.00 544.00 + 0.00 g382128019. 1.00 544.00 + 0.00 g3821280 20. 1.00 544.00 + 0.00 g3821280 21. 1.00544.00 + 0.00 g3821280 22. 1.00 544.00 + 0.00 g3821280 23. 1.00 544.00 +0.00 g3821280 24. 1.00 544.00 + 0.00 g3821280 25. 1.00 586.00 + 0.00g1351989 26. 1.00 586.00 + 0.00 g1351989 27. 1.00 586.00 + 0.00 g135198928. 1.00 586.00 + 0.00 g1351989 29. 1.00 586.00 + 0.00 g1351989 30. 1.00586.00 + 0.00 g1351989 31. 1.00 586.00 + 0.00 g1351989 32. 1.00 586.00 +0.00 g1351989 33. 1.00 586.00 + 0.00 g1351989 34. 1.00 586.00 + 0.00g1351989 35. 1.00 553.00 + 0.00 g399064 36. 1.00 586.00 + 0.00 g135198937. 1.00 544.00 + 0.00 g3821280 38. 1.00 586.00 + 0.00 g1351989 39. 1.00586.00 + 0.00 g1351989 40. 1.00 586.00 + 0.00 g1351989 41. 1.00 586.00 +0.00 g1351989 42. 1.00 586.00 + 0.00 g1351989 43. 1.00 586.00 + 0.00g1351989 44. 1.00 586.00 + 0.00 g1351989 45. 1.00 586.00 + 0.00 g135198946. 1.00 586.00 + 0.00 g1351989 47. 1.00 586.00 + 0.00 g1351989 48. 1.00544.00 + 0.00 g3821280 49. 1.00 568.00 + 1.0e−149 g6325403 50. 201.001226.00 + 3.0e−28  g6322209 51. 201.00 1226.00 + 3.0e−28  g6322209 52.1.00 568.00 + 1.0e−149 g6325403 53. 1.00 568.00 + 1.0e−149 g6325403 54.201.00 1226.00 + 3.0e−28  g6322209

FunCat Hit_Desc 1. Asparagine synthetase [glutamine-hydrolyzing](Glutamine- dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 2.asparagine synthetase type II [Phaseolus vulgaris] 3. asparaginesynthetase type II [Phaseolus vulgaris] 4. asparagine synthetase type II[Phaseolus vulgaris] 5. asparagine synthetase type II [Phaseolusvulgaris] 6. asparagine synthetase type II [Phaseolus vulgaris] 7.asparagine synthetase type II [Phaseolus vulgaris] 8. asparaginesynthetase type II [Phaseolus vulgaris] 9. asparagine synthetase type II[Phaseolus vulgaris] 10. asparagine synthetase type II [Phaseolusvulgaris] 11. asparagine synthetase type II [Phaseolus vulgaris] 12.Asparagine synthetase [glutamine-hydrolyzing] (Glutamine- dependentasparagine synthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 13. asparagine synthetase type II[Phaseolus vulgaris] 14. Asparagine synthetase [glutamine-hydrolyzing](Glutamine- dependent asparagine synthetase) gi|7438077|pir||T02978asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 15.asparagine synthetase type II [Phaseolus vulgaris] 16. asparaginesynthetase type II [Phaseolus vulgaris] 17. asparagine synthetase typeII [Phaseolus vulgaris] 18. asparagine synthetase type II [Phaseolusvulgaris] 19. asparagine synthetase type II [Phaseolus vulgaris] 20.asparagine synthetase type II [Phaseolus vulgaris] 21. asparaginesynthetase type II [Phaseolus vulgaris] 22. asparagine synthetase typeII [Phaseolus vulgaris] 23. asparagine synthetase type II [Phaseolusvulgaris] 24. asparagine synthetase type II [Phaseolus vulgaris] 25.Asparagine synthetase [glutamine-hydrolyzing] (Glutamine- dependentasparagine synthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 26. Asparagine synthetase[glutamine-hydrolyzing] (Glutamine- dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 27. Asparagine synthetase [glutamine-hydrolyzing] (Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978 asparaginesynthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 28.Asparagine synthetase [glutamine-hydrolyzing] (Glutamine- dependentasparagine synthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 29. Asparagine synthetase[glutamine-hydrolyzing] (Glutamine- dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 30. Asparagine synthetase [glutamine-hydrolyzing] (Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978 asparaginesynthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 31.Asparagine synthetase [glutamine-hydrolyzing] (Glutamine- dependentasparagine synthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 32. Asparagine synthetase[glutamine-hydrolyzing] (Glutamine- dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 33. Asparagine synthetase [glutamine-hydrolyzing] (Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978 asparaginesynthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 34.Asparagine synthetase [glutamine-hydrolyzing] (Glutamine- dependentasparagine synthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 35. Asparagine synthase[glutamine-hydrolyzing] (AS) gi|100980|pir||S25165 asparagine synthase(glutamine- hydrolysing) (EC 6.3.5.4) - garden asparagusgi|16076|emb|CAA48141.1|asparagine synthase (glutamine-hydrolysing)[Asparagus officinalis] 36. Asparagine synthetase[glutamine-hydrolyzing] (Glutamine- dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 37. asparagine synthetase type II [Phaseolus vulgaris] 38.Asparagine synthetase [glutamine-hydrolyzing] (Glutamine- dependentasparagine synthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 39. Asparagine synthetase[glutamine-hydrolyzing] (Glutamine- dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 40. Asparagine synthetase [glutamine-hydrolyzing] (Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978 asparaginesynthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 41.Asparagine synthetase [glutamine-hydrolyzing] (Glutamine- dependentasparagine synthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 42. Asparagine synthetase[glutamine-hydrolyzing] (Glutamine- dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 43. Asparagine synthetase [glutamine-hydrolyzing] (Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978 asparaginesynthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 44.Asparagine synthetase [glutamine-hydrolyzing] (Glutamine- dependentasparagine synthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 45. Asparagine synthetase[glutamine-hydrolyzing] (Glutamine- dependent asparagine synthetase)gi|7438077|pir||T02978 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - maize gi|984262|emb|CAA58052.1| asparragine synthetase [Zeamays] 46. Asparagine synthetase [glutamine-hydrolyzing] (Glutamine-dependent asparagine synthetase) gi|7438077|pir||T02978 asparaginesynthase (glutamine-hydrolysing) (EC 6.3.5.4) - maizegi|984262|emb|CAA58052.1| asparragine synthetase [Zea mays] 47.Asparagine synthetase [glutamine-hydrolyzing] (Glutamine- dependentasparagine synthetase) gi|7438077|pir||T02978 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - maize gi|984262|emb|CAA58052.1|asparragine synthetase [Zea mays] 48. asparagine synthetase type II[Phaseolus vulgaris] 49. Asn1p and Asn2p are isozymes; Asn1p[Saccharomyces cerevisiae] gi|1351984|sp|P49089|ASN1_YEAST Asparaginesynthetase [glutamine-hydrolyzing] 1 (Glutamine-dependent asparaginesynthetase 1) gi|1077018|pir||S52694 asparagine synthase(glutamine-hydrolysing) (EC 6.3.5.4) - yeast (Saccharomyces cerevisiae)gi|747902|emb|CAA88594.1| asparagine synthetase [Saccharomycescerevisiae] gi| 1066479|gb|AAB68284.1| Asn1p: Asparagine synthetase[Saccharomyces cerevisiae] 50. Required for invasion and pseudohyphaeformation in response to nitrogen starvation; Muc1p [Saccharomycescerevisiae] gi|728850|sp|P08640|AMYH_YEAST Glucoamylase S1/S2 precursor(Glucan 1,4-alpha-glucosidase) (1,4-alpha-D-glucan glucohydrolase)gi|626156|pir||S48478 glucan 1,4-alpha-glucosidase (EC 3.2.1.3) - yeast(Saccharomyces cerevisiae) gi|557822|emb| CAA86176.1| mal5, sta1, len:1367, CAI: 0.3, AMYH_YEAST P08640 GLUCOAMYLASE S1 (EC 3.2.1.3)[Saccharomyces cerevisiae] gi|1304387|gb|AAC49609.1| glucoamylase[Saccharomyces cerevisiae var. diastaticus] 51. Required for invasionand pseudohyphae formation in response to nitrogen starvation; Muc1p[Saccharomyces cerevisiae] gi|728850|sp|P08640|AMYH_YEAST GlucoamylaseS1/S2 precursor (Glucan 1,4-alpha-glucosidase) (1,4-alpha-D-glucanglucohydrolase) gi|626156|pir||S48478 glucan 1,4-alpha-glucosidase (EC3.2.1.3) - yeast (Saccharomyces cerevisiae) gi|557822|emb| CAA86176.1|mal5, sta1, len: 1367, CAI: 0.3, AMYH_YEAST P08640 GLUCOAMYLASE S1 (EC3.2.1.3) [Saccharomyces cerevisiae] gi|1304387|gb|AAC49609.1|glucoamylase [Saccharomyces cerevisiae var. diastaticus] 52. Asn1p andAsn2p are isozymes; Asn1p [Saccharomyces cerevisiae]gi|1351984|sp|P49089|ASN1_YEAST Asparagine synthetase[glutamine-hydrolyzing] 1 (Glutamine-dependent asparagine synthetase 1)gi|1077018|pir||S52694 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - yeast (Saccharomyces cerevisiae) gi|747902|emb|CAA88594.1|asparagine synthetase [Saccharomyces cerevisiae] gi|1066479|gb|AAB68284.1| Asn1p: Asparagine synthetase [Saccharomycescerevisiae] 53. Asn1p and Asn2p are isozymes; Asn1p [Saccharomycescerevisiae] gi|1351984|sp|P49089|ASN1_YEAST Asparagine synthetase[glutamine-hydrolyzing] 1 (Glutamine-dependent asparagine synthetase 1)gi|1077018|pir||S52694 asparagine synthase (glutamine-hydrolysing) (EC6.3.5.4) - yeast (Saccharomyces cerevisiae) gi|747902|emb|CAA88594.1|asparagine synthetase [Saccharomyces cerevisiae] gi|1066479|gb|AAB68284.1| Asn1p: Asparagine synthetase [Saccharomycescerevisiae] 54. Required for invasion and pseudohyphae formation inresponse to nitrogen starvation; Muc1p [Saccharomyces cerevisiae]gi|728850|sp|P08640|AMYH_YEAST Glucoamylase S1/S2 precursor (Glucan1,4-alpha-glucosidase) (1,4-alpha-D-glucan glucohydrolase)gi|626156|pir||S48478 glucan 1,4-alpha-glucosidase (EC 3.2.1.3) - yeast(Saccharomyces cerevisiae) gi|557822|emb| CAA86176.1| mal5, sta1, len:1367, CAI: 0.3, AMYH_YEAST P08640 GLUCOAMYLASE S1 (EC 3.2.1.3)[Saccharomyces cerevisiae] gi|1304387|gb|AAC49609.1| glucoamylase[Saccharomyces cerevisiae var. diastaticus]

FunCat_ FunCat_ FunCat_ FunCat_ FunCat_ FunCat_Hit_ Evalue Hit_NoPet_Iden Q_range Q_Coverage Coverage 1. 0.00 1.00 78.00 6.00 85.00100.00 2. 0.00 6.00 75.00 6.00 79.00 93.00 3. 0.00 6.00 75.00 6.00 79.0093.00 4. 0.00 6.00 75.00 6.00 79.00 93.00 5. 0.00 6.00 75.00 6.00 79.0093.00 6. 0.00 6.00 75.00 6.00 79.00 93.00 7. 0.00 6.00 75.00 6.00 79.0093.00 8. 0.00 6.00 75.00 6.00 79.00 93.00 9. 0.00 6.00 75.00 6.00 79.0093.00 10. 0.00 6.00 75.00 6.00 79.00 93.00 11. 0.00 6.00 75.00 6.0079.00 93.00 12. 0.00 1.00 78.00 6.00 85.00 100.00 13. 0.00 6.00 75.006.00 79.00 93.00 14. 0.00 1.00 78.00 6.00 85.00 100.00 15. 0.00 6.0075.00 6.00 79.00 93.00 16. 0.00 6.00 75.00 6.00 79.00 93.00 17. 0.006.00 75.00 6.00 79.00 93.00 18. 0.00 6.00 75.00 6.00 79.00 93.00 19.0.00 6.00 75.00 6.00 79.00 93.00 20. 0.00 6.00 75.00 6.00 79.00 93.0021. 0.00 6.00 75.00 6.00 79.00 93.00 22. 0.00 6.00 75.00 6.00 79.0093.00 23. 0.00 6.00 75.00 6.00 79.00 93.00 24. 0.00 6.00 75.00 6.0079.00 93.00 25. 0.00 1.00 78.00 6.00 85.00 100.00 26. 0.00 1.00 78.006.00 85.00 100.00 27. 0.00 1.00 78.00 6.00 85.00 100.00 28. 0.00 1.0078.00 6.00 85.00 100.00 29. 0.00 1.00 78.00 6.00 85.00 100.00 30. 0.001.00 78.00 6.00 85.00 100.00 31. 0.00 1.00 78.00 6.00 85.00 100.00 32.0.00 1.00 78.00 6.00 85.00 100.00 33. 0.00 1.00 78.00 6.00 85.00 100.0034. 0.00 1.00 78.00 6.00 85.00 100.00 35. 0.00 3.00 74.00 6.00 80.0094.00 36. 0.00 1.00 78.00 6.00 85.00 100.00 37. 0.00 6.00 75.00 6.0079.00 93.00 38. 0.00 1.00 78.00 6.00 85.00 100.00 39. 0.00 1.00 78.006.00 85.00 100.00 40. 0.00 1.00 78.00 6.00 85.00 100.00 41. 0.00 1.0078.00 6.00 85.00 100.00 42. 0.00 1.00 78.00 6.00 85.00 100.00 43. 0.001.00 78.00 6.00 85.00 100.00 44. 0.00 1.00 78.00 6.00 85.00 100.00 45.0.00 1.00 78.00 6.00 85.00 100.00 46. 0.00 1.00 78.00 6.00 85.00 100.0047. 0.00 1.00 78.00 6.00 85.00 100.00 48. 0.00 6.00 75.00 6.00 79.0093.00 49. 1.0e−149 64.00 48.00 6.00 79.00 99.00 50. 3.0e−28  148.0025.00 6.00 91.00 75.00 51. 3.0e−28  148.00 25.00 6.00 91.00 75.00 52.1.0e−149 64.00 48.00 6.00 79.00 99.00 53. 1.0e−149 64.00 48.00 6.0079.00 99.00 54. 3.0e−28  148.00 25.00 6.00 91.00 75.00

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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What is claimed is:
 1. A transgenic corn seed comprising in its genome aheterologous DNA construct comprising a promoter operably linked to apolynucleotide, the polynucleotide comprising: (a) a nucleic acidsequence comprising the sequence of SEQ ID NO:5; (b) a nucleic acidsequence encoding the polypeptide of SEQ ID NO:6; or (c) a nucleic acidsequence with at least 95% sequence identity to SEQ ID NO:5 that encodesan asparagine synthetase.
 2. The transgenic corn seed of claim 1,wherein said polynucleotide encodes SEQ ID NO:6.
 3. The transgenic cornseed of claim 1, wherein said polynucleotide comprises the nucleic acidsequence of SEQ ID NO:5.
 4. The transgenic corn seed of claim 1, whereinsaid promoter is a rice actin 1 promoter.
 5. A transgenic corn plantcomprising in its genome a heterologous DNA construct comprising apromoter operably linked to a polynucleotide, the polynucleotidecomprising: (a) a nucleic acid sequence comprising the sequence of SEQID NO:5; (b) a nucleic acid sequence encoding the polypeptide of SEQ IDNO:6; or (c) a nucleic acid sequence with at least 95% sequence identityto SEQ ID NO:5 that encodes an asparagine synthetase.
 6. The transgeniccorn plant of claim 5, wherein said polynucleotide encodes SEQ ID NO:6.7. The transgenic corn plant of claim 5, wherein said polynucleotidecomprises the sequence of SEQ ID NO:5.
 8. The transgenic corn plant ofclaim 5, wherein said promoter is a rice actin 1 promoter.